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Sample records for caspase-8 isoform caspase-8s

  1. The marine lipopeptide somocystinamide A triggers apoptosis via caspase 8

    OpenAIRE

    Wrasidlo, Wolf; Mielgo, Ainhoa; Torres, Vicente A; Barbero, Simone; Stoletov, Konstantin; Suyama, Takashi L.; Klemke, Richard L.; Gerwick, William H.; Carson, Dennis A.; Stupack, Dwayne G.

    2008-01-01

    Screening for novel anticancer drugs in chemical libraries isolated from marine organisms, we identified the lipopeptide somocystinamide A (ScA) as a pluripotent inhibitor of angiogenesis and tumor cell proliferation. The antiproliferative activity was largely attributable to induction of programmed cell death. Sensitivity to ScA was significantly increased among cells expressing caspase 8, whereas siRNA knockdown of caspase 8 increased survival after exposure to ScA. ScA rapidly and efficien...

  2. The human papillomavirus (HPV) E6 oncoproteins promotes nuclear localization of active caspase 8

    International Nuclear Information System (INIS)

    The HPV-16 E6 and E6⁎ proteins have been shown previously to be capable of regulating caspase 8 activity. We now show that the capacity of E6 to interact with caspase 8 is common to diverse HPV types, being also seen with HPV-11 E6, HPV-18 E6 and HPV-18 E6⁎. Unlike most E6-interacting partners, caspase 8 does not appear to be a major proteasomal target of E6, but instead E6 appears able to stimulate caspase 8 activation, without affecting the overall apoptotic activity. This would appear to be mediated in part by the ability of the HPV E6 oncoproteins to recruit active caspase 8 to the nucleus. - Highlights: • Multiple HPV E6 oncoproteins interact with the caspase 8 DED domain. • HPV E6 stimulates activation of caspase 8. • HPV E6 promotes nuclear accumulation of caspase 8

  3. The human papillomavirus (HPV) E6 oncoproteins promotes nuclear localization of active caspase 8

    Energy Technology Data Exchange (ETDEWEB)

    Manzo-Merino, Joaquin [Unidad de Investigación Biomédica en Cáncer, Instituto Nacional de Cancerología, México/Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México. Av. San Fernando No. 22, Col. Sección XVI, Tlalpan 14080 (Mexico); Massimi, Paola [International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34149 Trieste (Italy); Lizano, Marcela, E-mail: lizanosoberon@gmail.com [Unidad de Investigación Biomédica en Cáncer, Instituto Nacional de Cancerología, México/Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México. Av. San Fernando No. 22, Col. Sección XVI, Tlalpan 14080 (Mexico); Banks, Lawrence, E-mail: banks@icgeb.org [International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34149 Trieste (Italy)

    2014-02-15

    The HPV-16 E6 and E6{sup ⁎} proteins have been shown previously to be capable of regulating caspase 8 activity. We now show that the capacity of E6 to interact with caspase 8 is common to diverse HPV types, being also seen with HPV-11 E6, HPV-18 E6 and HPV-18 E6{sup ⁎}. Unlike most E6-interacting partners, caspase 8 does not appear to be a major proteasomal target of E6, but instead E6 appears able to stimulate caspase 8 activation, without affecting the overall apoptotic activity. This would appear to be mediated in part by the ability of the HPV E6 oncoproteins to recruit active caspase 8 to the nucleus. - Highlights: • Multiple HPV E6 oncoproteins interact with the caspase 8 DED domain. • HPV E6 stimulates activation of caspase 8. • HPV E6 promotes nuclear accumulation of caspase 8.

  4. EMT phenotype is induced by increased Src kinase activity via Src-mediated caspase-8 phosphorylation.

    Science.gov (United States)

    Zhao, Yang; Li, XiaoJun; Sun, XiangFei; Zhang, YunFeng; Ren, Hong

    2012-01-01

    Caspase-8 governs multiple cell responses to the microenvironmental cues. However, its integration of "death-life" signalings remains elusive. In our study, the role of caspase-8-Src is well-established as a promoter for migration or metastasis in Casp8(+)Src(+) A549/H226 cells in vivo and in vitro. In particular for nude mice models, mice implanted with Casp8(+)Src(+) A459/H226 cells remarkably increased spontaneous tumor metastatic burden with a significant survival disadvantage. Additionally, we detect that Src-mediated caspase-8 phosphorylation stimulates Src phosphorylation at Tyr-416 via the linkage of Src SH2 domain with phosph-Tyr-380 site of caspase-8. In turn, activated Src can efficiently induce epithelial-mesenchymal transition (EMT) phenotypic features to promote tumor cells metastasis. Surprisingly, RXDLL motif deletion in the DEDa of caspase-8 attenuates tumor cell migration or metastasis via impairing the recruitment of caspase-8 into the cellular periphery where activated Src is subject to caspase-8 phosphorylation. Together, a simple model is that the peripherization of caspase-8 is well-poised to facilitate Src-mediated caspase-8 phosphrylation at Tyr-380, then binding of phospho-Tyr380 of caspase-8 to Src SH2 domain may maintain Src in an active conformation to induce EMT phenotype, a key step toward cancer metastasis. PMID:22508042

  5. Caspase-8 Binding to Cardiolipin in Giant Unilamellar Vesicles Provides a Functional Docking Platform for Bid

    DEFF Research Database (Denmark)

    Jalmar, Olivier; Franc¸ois-Moutal, Liberty; García-Sáez, Ana-Jesus;

    2013-01-01

    Caspase-8 is involved in death receptor-mediated apoptosis in type II cells, the proapoptotic programme of which is triggered by truncated Bid. Indeed, caspase-8 and Bid are the known intermediates of this signalling pathway. Cardiolipin has been shown to provide an anchor and an essential activa...

  6. Novel HTS strategy identifies TRAIL-sensitizing compounds acting specifically through the caspase-8 apoptotic axis.

    Directory of Open Access Journals (Sweden)

    Darren Finlay

    Full Text Available Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL is potentially a very important therapeutic as it shows selectivity for inducing apoptosis in cancer cells whilst normal cells are refractory. TRAIL binding to its cognate receptors, Death Receptors-4 and -5, leads to recruitment of caspase-8 and classical activation of downstream effector caspases, leading to apoptosis. As with many drugs however, TRAIL's usefulness is limited by resistance, either innate or acquired. We describe here the development of a novel 384-well high-throughput screening (HTS strategy for identifying potential TRAIL-sensitizing agents that act solely in a caspase-8 dependent manner. By utilizing a TRAIL resistant cell line lacking caspase-8 (NB7 compared to the same cells reconstituted with the wild-type protein, or with a catalytically inactive point mutant of caspase-8, we are able to identify compounds that act specifically through the caspase-8 axis, rather than through general toxicity. In addition, false positive hits can easily be "weeded out" in this assay due to their activity in cells lacking caspase-8-inducible activity. Screening of the library of pharmacologically active compounds (LOPAC was performed as both proof-of-concept and to discover potential unknown TRAIL sensitizers whose mechanism is caspase-8 mediated. We identified known TRAIL sensitizers from the library and identified new compounds that appear to sensitize specifically through caspase-8. In sum, we demonstrate proof-of-concept and discovery of novel compounds with a screening strategy optimized for the detection of caspase-8 pathway-specific TRAIL sensitizers. This screen was performed in the 384-well format, but could easily be further miniaturized, allows easy identification of artifactual false positives, and is highly scalable to accommodate diverse libraries.

  7. A synthetic lethal screen identifies FAT1 as an antagonist of caspase-8 in extrinsic apoptosis

    OpenAIRE

    Kranz, Dominique; Boutros, Michael

    2014-01-01

    The extrinsic apoptosis pathway is initiated by binding of death ligands to death receptors resulting in the formation of the death-inducing signaling complex (DISC). Activation of procaspase-8 within the DISC and its release from the signaling complex is required for processing executor caspases and commiting cell death. Here, we report that the atypical cadherin FAT1 interacts with caspase-8 preventing the association of caspase-8 with the DISC. We identified FAT1 in a genome-wide siRNA scr...

  8. Effects of methylation status of caspase-8 promoter on antitumor activity of TRAIL to human gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG Ru-gang; FANG Dian-chun; YANG Liu-qin; LUO Yuan-gang

    2004-01-01

    Objective: To study the effects of the methylation status of caspase-8 promoter on the antitumor activity of TRAIL to the human gastric cancer cells. Methods: The methylation of caspase-8 was measured with methylation specific PCR (MSP) and the antitomor capability of TRAIL to human gastric cancer cells was determined with MTT. Results: No methylation of caspase-8 in the human gastric cancer cells was found. The sensitivity of 5 lines of gastric cancer cells to the antitumor activity of TRAIL was different. The administration of the demethylation agent 5-Aza-2'-deoxycytidine ( 5-AzaCdR) increased the sensitivity of gastric cancer cells to TRAIL but did not change the methylation status of caspase-8 promoter in gastric cancer cells. Conclusion: 5-Aza-CdR increases the sensitivity of most of gastric cancer cells to TRAIL but caspase-8 is not involved in the antitumor activity of TRAIL.

  9. c-FLIP及Caspase-8表达在乳腺癌组织中的表达及临床意义%Expression of c-FLIP and Caspase-8 in Breast Cancer and Their Clinical Significance

    Institute of Scientific and Technical Information of China (English)

    潘峥; 陈军; 覃冠德; 覃思繁

    2012-01-01

    Objective To investigate the expression of c-FLIP and Caspase-8 in breast cancer and their correlation with metastasis and recurrence. Methods Immunohistochemisty was employed to determine the expression of c-FLIP and Caspase-8 in 80 breast carcinomas and paired adjacent breast tissues. Results c-FLIP was overexpressed in breast cancers compared with paired adjacent breast tissues. The expression of c-FLIP was significantly correlated with TNM stages, histological grade, axillary lymph node metastasis, the recurrence of tumor and the express of C-erbB-2. The expression of Caspase-8 was significantly lower in breast cancers compared with paired adjacent breast tissues. The expression of Caspase-8 was significantly correlated with histological grade, tumor size. There was a significantly negative relationship of c-FLIP with Caspase-8. Conclusion The overexpres-sion of c-FLIP and the inactivation of Caspase-8 might enhance the tumour cell proliferation and malignance in breast cancer.%目的 探讨凋亡相关蛋白c-FLIP和Caspase-8在人乳腺癌组织中的表达及相关性,分析其与乳腺癌转移复发的关系.方法 应用免疫组织化学法检测80例乳腺癌组织和相应的癌旁组织中c-FLIP和Caspase-8的表达情况.结果 80例乳腺癌组织中c-FLIP表达的阳性率显著高于相应的癌旁组织(P<0.05),c-FLIP的表达与TNM分期、组织学分级、腋窝淋巴结转移、术后复发及C-erbB-2表达密切相关(P<0.05);Caspase-8表达的阳性率显著低于相应的癌旁组织(P<0.05),Caspase-8的表达与组织学分级、肿瘤大小密切相关(P<0.05).c-FLIP表达和Caspase-8表达有显著负相关性(γ=-0.380,P=0.001).结论 c-FLIP的高表达及Caspase-8表达缺失可能在乳腺癌发生发展中起重要作用.

  10. Caspase-8 regulates TNF-α-induced epithelial necroptosis and terminal ileitis.

    Science.gov (United States)

    Günther, Claudia; Martini, Eva; Wittkopf, Nadine; Amann, Kerstin; Weigmann, Benno; Neumann, Helmut; Waldner, Maximilian J; Hedrick, Stephen M; Tenzer, Stefan; Neurath, Markus F; Becker, Christoph

    2011-09-15

    Dysfunction of the intestinal epithelium is believed to result in the excessive translocation of commensal bacteria into the bowel wall that drives chronic mucosal inflammation in Crohn's disease, an incurable inflammatory bowel disease in humans characterized by inflammation of the terminal ileum. In healthy individuals, the intestinal epithelium maintains a physical barrier, established by the tight contact of cells. Moreover, specialized epithelial cells such as Paneth cells and goblet cells provide innate immune defence functions by secreting mucus and antimicrobial peptides, which hamper access and survival of bacteria adjacent to the epithelium. Epithelial cell death is a hallmark of intestinal inflammation and has been discussed as a possible pathogenic mechanism driving Crohn's disease in humans. However, the regulation of epithelial cell death and its role in intestinal homeostasis remain poorly understood. Here we demonstrate a critical role for caspase-8 in regulating necroptosis of intestinal epithelial cells (IECs) and terminal ileitis. Mice with a conditional deletion of caspase-8 in the intestinal epithelium (Casp8(ΔIEC)) spontaneously developed inflammatory lesions in the terminal ileum and were highly susceptible to colitis. Casp8(ΔIEC) mice lacked Paneth cells and showed reduced numbers of goblet cells, indicating dysregulated antimicrobial immune cell functions of the intestinal epithelium. Casp8(ΔIEC) mice showed increased cell death in the Paneth cell area of small intestinal crypts. Epithelial cell death was induced by tumour necrosis factor (TNF)-α, was associated with increased expression of receptor-interacting protein 3 (Rip3; also known as Ripk3) and could be inhibited on blockade of necroptosis. Lastly, we identified high levels of RIP3 in human Paneth cells and increased necroptosis in the terminal ileum of patients with Crohn's disease, suggesting a potential role of necroptosis in the pathogenesis of this disease. Together, our

  11. Voltage dependent anion channel-1 regulates death receptor mediated apoptosis by enabling cleavage of caspase-8

    International Nuclear Information System (INIS)

    Activation of the extrinsic apoptosis pathway by tumour necrosis factor related apoptosis inducing ligand (TRAIL) is a novel therapeutic strategy for treating cancer that is currently under clinical evaluation. Identification of molecular biomarkers of resistance is likely to play an important role in predicting clinical anti tumour activity. The involvement of the mitochondrial type 1 voltage dependent anion channel (VDAC1) in regulating apoptosis has been highly debated. To date, a functional role in regulating the extrinsic apoptosis pathway has not been formally excluded. We carried out stable and transient RNAi knockdowns of VDAC1 in non-small cell lung cancer cells, and stimulated the extrinsic apoptotic pathway principally by incubating cells with the death ligand TRAIL. We used in-vitro apoptotic and cell viability assays, as well as western blot for markers of apoptosis, to demonstrate that TRAIL-induced toxicity is VDAC1 dependant. Confocal microscopy and mitochondrial fractionation were used to determine the importance of mitochondria for caspase-8 activation. Here we show that either stable or transient knockdown of VDAC1 is sufficient to antagonize TRAIL mediated apoptosis in non-small cell lung cancer (NSCLC) cells. Specifically, VDAC1 is required for processing of procaspase-8 to its fully active p18 form at the mitochondria. Loss of VDAC1 does not alter mitochondrial sensitivity to exogenous caspase-8-cleaved BID induced mitochondrial depolarization, even though VDAC1 expression is essential for TRAIL dependent activation of the intrinsic apoptosis pathway. Furthermore, expression of exogenous VDAC1 restores the apoptotic response to TRAIL in cells in which endogenous VDAC1 has been selectively silenced. Expression of VDAC1 is required for full processing and activation of caspase-8 and supports a role for mitochondria in regulating apoptosis signaling via the death receptor pathway

  12. TRAF2 Sets a threshold for extrinsic apoptosis by tagging caspase-8 with a ubiquitin shutoff timer.

    Science.gov (United States)

    Gonzalvez, Francois; Lawrence, David; Yang, Becky; Yee, Sharon; Pitti, Robert; Marsters, Scot; Pham, Victoria C; Stephan, Jean-Philippe; Lill, Jennie; Ashkenazi, Avi

    2012-12-28

    Apoptotic caspase activation mechanisms are well defined, yet inactivation modes remain unclear. The death receptors (DRs), DR4, DR5, and Fas, transduce cell-extrinsic apoptotic signals by recruiting caspase-8 into a death-inducing signaling complex (DISC). At the DISC, Cullin3-dependent polyubiquitination on the small catalytic subunit of caspase-8 augments stimulation. Here we report that tumor necrosis factor receptor-associated factor 2 (TRAF2) interacts with caspase-8 at the DISC, downstream of Cullin3. TRAF2 directly mediates RING-dependent, K48-linked polyubiquitination on the large catalytic domain of caspase-8. This modification destines activated caspase-8 molecules to rapid proteasomal degradation upon autoprocessing and cytoplasmic translocation. TRAF2 depletion lowers the signal threshold for DR-mediated apoptosis, altering cell life versus death decisions in vitro and in vivo. Thus, TRAF2 sets a critical barrier for cell-extrinsic apoptosis commitment by tagging activated caspase-8 with a K48-ubiquitin shutoff timer. These results may have important implications for caspase regulation mechanisms.

  13. The Caspase-8 Homolog Dredd Cleaves Imd and Relish but Is Not Inhibited by p35*

    Science.gov (United States)

    Kim, Chan-Hee; Paik, Donggi; Rus, Florentina; Silverman, Neal

    2014-01-01

    In Drosophila, the Imd pathway is activated by diaminopimelic acid-type peptidoglycan and triggers the humoral innate immune response, including the robust induction of antimicrobial peptide gene expression. Imd and Relish, two essential components of this pathway, are both endoproteolytically cleaved upon immune stimulation. Genetic analyses have shown that these cleavage events are dependent on the caspase-8 like Dredd, suggesting that Imd and Relish are direct substrates of Dredd. Among the seven Drosophila caspases, we find that Dredd uniquely promotes Imd and Relish processing, and purified recombinant Dredd cleaves Imd and Relish in vitro. In addition, interdomain cleavage of Dredd is not required for Imd or Relish processing and is not observed during immune stimulation. Baculovirus p35, a suicide substrate of executioner caspases, is not cleaved by purified Dredd in vitro. Consistent with this biochemistry but contrary to earlier reports, p35 does not interfere with Imd signaling in S2* cells or in vivo. PMID:24891502

  14. Ofloxacin induces apoptosis in microencapsulated juvenile rabbit chondrocytes by caspase-8-dependent mitochondrial pathway

    International Nuclear Information System (INIS)

    Quinolones (QNs)-induced arthropathy is an important toxic effect in immature animals leading to restriction of their therapeutic use in pediatrics. However, the exact mechanism still remains unclear. Recently, we have demonstrated that ofloxacin, a typical QN, induces apoptosis of alginate microencapsulated juvenile rabbit joint chondrocytes by disturbing the β1 integrin functions and inactivating the ERK/MAPK signaling pathway. In this study, we extend our initial observations to further elucidate the mechanism(s) of ofloxacin-induced apoptosis by utilizing specific caspase inhibitors. Pretreatment with both caspase-9-specific inhibitor zLEHD-fmk and caspase-8 inhibitor zIETD-fmk attenuated ofloxacin-induced apoptosis and activation of caspase-3 of chondrocyte in a concentration-dependent manner, as determined by fluorescent dye staining, enzyme activity assay and immunoblotting. Furthermore, the activation of caspase-9, -8 and -3 stimulated by ofloxacin was significantly inhibited in the presence of zIETD-fmk while pretreatment with zLEHD-fmk only blocked the activation of caspase-9 and -3. Ofloxacin also stimulated a concentration-dependent translocation of cytochrome c from mitochondria into the cytosol and a decrease of mitochondrial transmembrane potential, which was completely inhibited by zIETD-fmk. In addition, ofloxacin was found to increase the level of Bax, tBid, p53 in a concentration- and time-dependent manner. Taken together, The current results indicate that the caspase-8-dependent mitochondrial pathway is primarily involved in the ofloxacin-induced apoptosis of microencapsulated juvenile rabbit joint chondrocytes

  15. Molecular cloning, immunohistochemical localization, characterization and expression analysis of caspase-8 from the blunt snout bream (Megalobrama amblycephala) exposed to ammonia.

    Science.gov (United States)

    Sun, Shengming; Ge, Xianping; Zhu, Jian; Zhang, Wuxiao; Zhang, Qiong

    2015-12-01

    Caspase-8 is an initiator caspase that plays a crucial role in some cases of apoptosis by extrinsic and intrinsic pathways. Caspase-8 structure and function have been extensively studied in mammals, but in fish the characterization of that initiator caspase is still scarce. In this study, we isolated the caspase-8 gene from Megalobrama amblycephala, one of the most important industrial aquatic animals in China using rapid amplification of cDNA ends (RACE). The 2034 bp full-length M. amblycephala caspase-8 cDNA sequence contained an ORF of 1467 bp encoding a polypeptide of 489 amino acid residues, a 5'-UTR of 102 bp and a 3'-UTR of 462 bp. The caspase-8 amino acid sequences contained two highly conservative death effector domains (DEDs) at N-terminal, the caspase family domains P20 and P10, caspase-8 active-site pentapeptide and potential aspartic acid cleavage sites. Phylogenetic analysis revealed that M. amblycephala caspase-8 were clustered with the caspase-8 from other vertebrate. Real-time quantitative PCR analysis revealed that caspase-8 transcripts were detected in liver after exposure to ammonia. Meanwhile using Western blot analysis, caspase-8 cleaved fragment was detected and significant alteration of procaspase-8 level was found with the same ammonia treatment condition. Furthermore, the result of immunohistochemical detection showed that remarkable changes of immunopositive staining were observed after ammonia treatment. Accordingly, the results signify that caspase-8 of fish may play an essential role in ammonia induced apoptosis.

  16. Caspase-8 activity is part of the BeWo trophoblast cell defense mechanisms against Trypanosoma cruzi infection.

    Science.gov (United States)

    Carrillo, Ileana; Droguett, Daniel; Castillo, Christian; Liempi, Ana; Muñoz, Lorena; Maya, Juan Diego; Galanti, Norbel; Kemmerling, Ulrike

    2016-09-01

    Congenital Chagas disease is caused by the protozoan parasite Trypanosoma cruzi that must cross the placental barrier during transmission. The trophoblast constitutes the first tissue in contact with the maternal-blood circulating parasite. Importantly, the congenital transmission rates are low, suggesting the presence of local placental defense mechanisms. Cellular proliferation and differentiation as well as apoptotic cell death are induced by the parasite and constitute part of the epithelial turnover of the trophoblast, which has been suggested to be part of those placental defenses. On the other hand, caspase-8 is an essential molecule in the modulation of trophoblast turnover by apoptosis and by epithelial differentiation. As an approach to study whether T. cruzi induced trophoblast turnover and infection is mediated by caspase-8, we infected BeWo cells (a trophoblastic cell line) with the parasite and determined in the infected cells the expression and enzymatic activity of caspase-8, DNA synthesis (as proliferation marker), β-human chorionic gonadotropin (β-hCG) (as differentiation marker) and activity of Caspase-3 (as apoptotic death marker). Parasite load in BeWo cells was measured by DNA quantification using qPCR and cell counting. Our results show that T. cruzi induces caspase-8 activity and that its inhibition increases trophoblast cells infection while decreases parasite induced cellular differentiation and apoptotic cell death, but not cellular proliferation. Thus, caspase-8 activity is part of the BeWo trophoblast cell defense mechanisms against T. cruzi infection. Together with our previous results, we suggest that the trophoblast turnover is part of local placental anti-parasite mechanisms. PMID:27328973

  17. Molecular and acute temperature stress response characterizations of caspase-8 gene in two mussels, Mytilus coruscus and Mytilus galloprovincialis.

    Science.gov (United States)

    Zhang, Duo; Wang, Hong-Wei; Yao, Cui-Luan

    2014-01-01

    The caspase family represents aspartate-specific cysteine proteases that play key roles in initiation of apoptosis in various cells response to environmental stress. In this study, two caspase-8 cDNA sequences were cloned from two Mytilus mussels, Mytilus coruscus (Mccaspase-8) and Mytilus galloprovincialis (Mgcaspase-8), respectively. The full-length cDNA of Mccaspase-8 was 1884bp, including a 5'-terminal untranslated region (UTR) of 140bp, a 3'-terminal UTR of 238bp and an open reading frame (ORF) of 1506bp encoding a polypeptide of 501 amino acids. The 1775bp full-length Mg caspase-8 cDNA sequence contained an ORF of 1488bp encoding a polypeptide of 495 amino acid residues, a 5'-UTR of 51bp and a 3'-UTR of 236bp. Both the Mccaspase-8 and Mgcaspase-8 amino acid sequences contained two highly conservative death effector domains (DEDs) at N-terminal, the caspase family domains P20 and P10 and the caspase family cysteine active site 'KPKLFFIQACQG'. Phylogenetic analysis revealed that Mccaspase-8 and Mgcaspase-8 were clustered with the caspase-8 from other organisms, with the close relationship with caspase-8 from mollusk. Quantitative real-time reverse transcription PCR (qRT-PCR) analysis indicated that the predominant transcripts of Mccaspase-8 were in mantle and gonad tissue of M. coruscus and the high expression levels of Mgcaspase-8 were in digestive gland and gill tissue of M. galloprovincialis, respectively. The impacts of temperature stress on Mccaspase-8 and Mgcaspase-8 expressions were tested in gill tissue and hemocytes of both species. Our results showed that both Mccaspase-8 and Mgcaspase-8 transcripts and caspase-8 activity in gill tissue and hemocytes could be induced significantly after cold and heat stress (pgalloprovincialis.

  18. Induction of Caspase 8 by 5-Azacytidine renders NB cells sensitive to TRAIL%5氮杂胞苷诱导NB细胞Caspase 8表达及对TRAIL敏感性的研究

    Institute of Scientific and Technical Information of China (English)

    佟海侠; 张锦华; 张继红; 陆春伟

    2005-01-01

    目的:探讨5氮杂胞苷(5-Azacytidine)对肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor related apoptosis inducing ligand,TRAIL)诱导神经母细胞瘤(neuroblastoma,NB)细胞株SH-SY5Y凋亡的影响及其发生机制.方法:应用RT-PCR方法检测5氮杂胞苷作用前后SY5Y细胞Caspase 8的表达;应用四甲基偶氮唑蓝(MTT)比色法及流式细胞仪(FCM)检测TRAIL、5氮杂胞苷+TRAIL、5氮杂胞苷+Caspase 8抑制剂+TRAIL对SY5Y细胞生长及凋亡的影响.结果:SY5Y细胞不表达Caspase 8,5氮杂胞苷作用1、3和5d的SY5Y细胞Caspase 8表达逐渐增加;SY5Y细胞对TRAIL不敏感,经5氮杂胞苷诱导表达Caspase 8的 SY5Y 细胞对TRAIL敏感,并呈现一定的时间和剂量依赖性.联合用药组细胞凋亡率分别为(28.6±4.0)%、(38.5±4.2)%和(42.3±6.2)%,与单独用TRAIL组[(9.5±2.0)%、(10.7±1.8)%和(11.2±1.5)%]比较,差异有统计学意义,F值分别为41.131、45.014和49.405,P值均为0.000;Caspase 8抑制剂组细胞凋亡率分别为(11.8±2.0)%、(13.5±2.9)%和(15.1±3.4)%,与相应浓度的5氮杂胞苷+TRAIL组相比,细胞凋亡率明显降低,F值分别为41.131、45.014和49.405,P值均为0.000,说明Caspase 8抑制剂可明显降低TRAIL对表达Caspase 8的SY5Y细胞的杀伤作用.结论:5氮杂胞苷能增强SY5Y细胞对TRAIL的敏感性,其发生机制可能与Caspase 8表达上调有关.

  19. Radiation-Inducible Caspase-8 Gene Therapy for Malignant Brain Tumors

    International Nuclear Information System (INIS)

    Purpose: Patients with malignant gliomas have a poor prognosis. To explore a novel and more effective approach for the treatment of patients with malignant gliomas, we designed a strategy that combines caspase-8 (CSP8) gene therapy and radiation treatment (RT). In addition, the specificity of the combined therapy was investigated to decrease the unpleasant effects experienced by the surrounding normal tissue. Methods and Materials: We constructed the plasmid pEGR-green fluorescence protein that included the radiation-inducible early growth response gene-1 (Egr-1) promoter and evaluated its characteristics. The pEGR-CSP8 was constructed and included the Egr-1 promoter and CSP8 complementary DNA. Assays that evaluated the apoptosis inducibility and cytotoxicity caused by CSP8 gene therapy combined with RT were performed using U251 and U87 glioma cells. The pEGR-CSP8 was transfected into the subcutaneous U251 glioma cells of nude mice by means of in vivo electroporation. The in vivo effects of CSP8 gene therapy combined with RT were evaluated. Results: The Egr-1 promoter yielded a better response with fractionated RT than with single-dose RT. In the assay of apoptosis inducibility and cytotoxicity, pEGR-CSP8 showed response for RT. The pEGR-CSP8 combined with RT is capable of inducing cell death effectively. In mice treated with pEGR-CSP8 and RT, apoptotic cells were detected in pathologic sections, and a significant difference was observed in tumor volumes. Conclusions: Our results indicate that radiation-inducible gene therapy may have great potential because this can be spatially or temporally controlled by exogenous RT and is safe and specific

  20. Fatal Hepatitis Mediated by TNFα Requires Caspase-8 and Involves the BH3-only Proteins Bid and Bim

    Science.gov (United States)

    Kaufmann, Thomas; Jost, Philipp J; Pellegrini, Marc; Puthalakath, Hamsa; Gugasyan, Raffi; Gerondakis, Steve; Cretney, Erika; Smyth, Mark J; Silke, John; Hakem, Razq; Bouillet, Philippe; Mak, Tak W; Dixit, Vishva M; Strasser, Andreas

    2009-01-01

    SUMMARY Apoptotic death of hepatocytes, a feature and contributing factor of many chronic and acute liver diseases, can be a consequence of over-activation of the immune system. Injection with lipopolysaccharide (LPS) plus the transcriptional inhibitor D(+)-galactosamine (GalN) or mitogenic T cell activation cause fatal hepatocyte apoptosis in mice. In both settings hepatocyte killing is mediated by TNFα/TNF-R signaling but the effector mechanisms remain unclear. Our analysis of gene-targeted mice showed that caspase-8 is essential for hepatocyte killing in both settings. Loss of Bid, the pro-apoptotic BH3-only protein activated by caspase-8, and essential for Fas ligand-induced hepatocyte killing, resulted only in a minor reduction of liver damage. However, combined loss of Bid and another BH3-only protein, Bim, activated by JNK, protected mice from LPS+GalN-induced hepatitis. These observations identify caspase-8 and the BH3-only proteins Bid and Bim as potential therapeutic targets for treatment of inflammatory liver diseases. PMID:19119023

  1. Diatom-derived oxylipins induce cell death in sea urchin embryos activating caspase-8 and caspase 3/7.

    Science.gov (United States)

    Ruocco, Nadia; Varrella, Stefano; Romano, Giovanna; Ianora, Adrianna; Bentley, Matt G; Somma, Domenico; Leonardi, Antonio; Mellone, Stefano; Zuppa, Antonio; Costantini, Maria

    2016-07-01

    Diatoms are an important class of unicellular algae that produce bioactive secondary metabolites with cytotoxic activity collectively termed oxylipins, including polyunsaturated aldehydes (PUAs), hydroxyacids (HEPEs), oxo-acids and epoxyalcohols. Previous results showed that at higher concentrations, the PUA decadienal induced apoptosis on copepods and sea urchin embryos via caspase-3 activation; at lower concentrations decadienal affected the expression levels of the caspase-8 gene in embryos of the sea urchin Paracentrotus lividus. In the present work, we studied the effects of other common oxylipins produced by diatoms: two PUAs (heptadienal and octadienal) and four hydroxyacids (5-, 9- 11- and 15-HEPE) on P. lividus cell death and caspase activities. Our results showed that (i) at higher concentrations PUAs and HEPEs induced apoptosis in sea urchin embryos, detected by microscopic observation and through the activation of caspase-3/7 and caspase-8 measured by luminescent assays; (ii) at low concentrations, PUAs and HEPEs affected the expression levels of caspase-8 and caspase-3/7 (isolated for the first time here in P. lividus) genes, detected by Real Time qPCR. These findings have interesting implications from the ecological point of view, given the importance of diatom blooms in nutrient-rich aquatic environments. PMID:27130972

  2. Induction of caspase 8 and reactive oxygen species by ruthenium-derived anticancer compounds with improved water solubility and cytotoxicity.

    Science.gov (United States)

    Vidimar, Vania; Meng, Xiangjun; Klajner, Marcelina; Licona, Cynthia; Fetzer, Ludivine; Harlepp, Sébastien; Hébraud, Pascal; Sidhoum, Marjorie; Sirlin, Claude; Loeffler, Jean-Philippe; Mellitzer, Georg; Sava, Gianni; Pfeffer, Michel; Gaiddon, Christian

    2012-12-01

    Organometallic compounds which contain metals, such as ruthenium or gold, have been investigated as a replacement for platinum-derived anticancer drugs. They often show good antitumor effects, but the identification of their precise mode of action or their pharmacological optimization is still challenging. We have previously described a class of ruthenium(II) compounds with interesting anticancer properties. In comparison to cisplatin, these molecules have lower side effects, a reduced ability to interact with DNA, and they induce cell death in absence of p53 through CHOP/DDIT3. We have now optimized these molecules by improving their cytotoxicity and their water solubility. In this article, we demonstrate that by changing the ligands around the ruthenium we modify the ability of the compounds to interact with DNA. We show that these optimized molecules reduce tumor growth in different mouse models and retain their ability to induce CHOP/DDIT3. However, they are more potent inducers of cancer cell death and trigger the production of reactive oxygen species and the activation of caspase 8. More importantly, we show that blocking reactive oxygen species production or caspase 8 activity reduces significantly the activity of the compounds. Altogether our data suggest that water-soluble ruthenium(II)-derived compounds represent an interesting class of molecules that, depending on their structures, can target several pro-apoptotic signaling pathways leading to reactive oxygen species production and caspase 8 activation. PMID:22964219

  3. Dasatinib promotes paclitaxel-induced necroptosis in lung adenocarcinoma with phosphorylated caspase-8 by c-Src.

    Science.gov (United States)

    Diao, Yan; Ma, Xiaobin; Min, WeiLi; Lin, Shuai; Kang, HuaFeng; Dai, ZhiJun; Wang, Xijing; Zhao, Yang

    2016-08-28

    Cisplatin and paclitaxel are considered to be the backbone of chemotherapy in lung adenocarcinoma. These agents show pleiotropic effects on cell death. However, the precise mechanisms remain unclear. The present study reported that phosphorylated caspase-8 at tyrosine 380 (p-Casp8) was characterized as a biomarker of chemoresistance to TP regimen (cisplatin and paclitaxel) in patients with resectable lung adenocarcinoma with significantly poorer 5-year disease-free survival (DFS) and overall survival (OS). Cisplatin killed lung adenocarcinoma cells regardless of c-Src-induced caspase-8 phosphorylation at tyrosine 380. Subsequently, we identified a novel mechanism by which paclitaxel induced necroptosis in lung adenocarcinoma cells that was dependent upon p-Casp8, receptor-interacting protein kinase 1 (RIPK1), and RIPK3. Moreover, dasatinib, a c-Src inhibitor, dephosphorylated caspase-8 to facilitate necroptosis, rather than apoptosis, in paclitaxel-treated p-Casp8-expressing lung adenocarcinoma cells. The data from our study revealed previously unrecognized roles of p-Casp8 as a positive effector in the initiation of necroptosis and as a negative effector in the repression of the interaction between RIPK1 and RIPK3. Moreover, these outcomes supported the need for further clinical studies with the goal of evaluating the efficacy of dasatinib plus paclitaxel in the treatment of lung adenocarcinoma. PMID:27195913

  4. RIP3 Inhibits Inflammatory Hepatocarcinogenesis but Promotes Cholestasis by Controlling Caspase-8- and JNK-Dependent Compensatory Cell Proliferation

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    Mihael Vucur

    2013-08-01

    Full Text Available For years, the term “apoptosis” was used synonymously with programmed cell death. However, it was recently discovered that receptor interacting protein 3 (RIP3-dependent “necroptosis” represents an alternative programmed cell death pathway activated in many inflamed tissues. Here, we show in a genetic model of chronic hepatic inflammation that activation of RIP3 limits immune responses and compensatory proliferation of liver parenchymal cells (LPC by inhibiting Caspase-8-dependent activation of Jun-(N-terminal kinase in LPC and nonparenchymal liver cells. In this way, RIP3 inhibits intrahepatic tumor growth and impedes the Caspase-8-dependent establishment of specific chromosomal aberrations that mediate resistance to tumor-necrosis-factor-induced apoptosis and underlie hepatocarcinogenesis. Moreover, RIP3 promotes the development of jaundice and cholestasis, because its activation suppresses compensatory proliferation of cholangiocytes and hepatic stem cells. These findings demonstrate a function of RIP3 in regulating carcinogenesis and cholestasis. Controlling RIP3 or Caspase-8 might represent a chemopreventive or therapeutic strategy against hepatocellular carcinoma and biliary disease.

  5. 5氮杂胞苷联合干扰素-γ上调神经母细胞瘤细胞Caspase 8表达的研究%5-Azacytidine and IFN-gamma cooperate to upregulate Caspase-8 expression in neuroblastoma cells

    Institute of Scientific and Technical Information of China (English)

    佟海侠; 陆春伟; 王秋实; 王立维

    2011-01-01

    Objective : To investigate if induction of Caspase 8 by demethylation agent 5 - Azacytidine( 5AZA ) and/or γ - interferon ( IFNγ ) renders neuroblastoma ( NB ) cells sensitive to tumor necrosis factor related apoptosis inducing ligand( TRAIL ). Methods: Caspase 8 expression was monitored by Western blot analysis. The effects of 5AZA,IFNγ, TRAIL, 5AZA and/or IFNγ +TRAIL, 5AZA and/or IFNγ + Caspase 8 inhibitor + TRAIL on the growth and apoptosis of NB cells were detected with flow cytometry. The relative Caspase 8 activity was measured with colorimetric assay. Results : Caspase 8 was undetectable in SH - SY5Y( SY5Y ) cells which were resistant to TRAIL , but an increased expression of Caspase 8 was found after treatment with 5AZA and/or IFNγ. The level of Caspase 8 protein in IFNγ +5AZA group was higher than that in single treatment group. Moreover, TRAIL combined with 5AZA and/or IFNγ induced apoptosis in SY5Y cells. The relative Caspase 8 activity of SY5Y cells in SAZA and/or IFNγ + TRAIL group was significantly higher than those of control group, 5AZA group, IFNγ group, TRAIL group and Caspase 8 inhibitor group. Conclusion :5 - AZA and IFNγ could cooperate to upregulate Caspase - 8 expression in NB cells. TRAIL induced apoptosis in NB cells expressing Caspase 8 with an increase of relative Caspase 8 activity.%目的:应用去甲基药5氮杂胞苷(5AZA)和/或干扰素(IFNγ)诱导神经母细胞瘤(neuroblastoma,NB)细胞Caspase 8的表达,并观察是否可以恢复NB细胞对肿瘤坏死因子相关凋亡诱导配体(TRAIL)的敏感性.方法:应用Western blot方法检测5AZA和/或IFNγ作用前后SY5Y细胞Caspase 8蛋白的表达;应用流式细胞术检测5AZA 、IFNγ、TRAIL、5AZA 和/或IFNγ+TRAIL、5AZA和/或IFNγ+Caspase 8抑制剂zIETD-FMK+TRAIL对NB细胞生长及凋亡的影响;应用比色法测定Caspase 8相对活性.结果:对TRAIL耐药的SY5Y细胞不表达Caspase 8,经5AZA 和/或IFNγ处理后Caspase 8 蛋白

  6. Analysis of death receptor 5 and caspase-8 expression in primary and metastatic head and neck squamous cell carcinoma and their prognostic impact.

    Directory of Open Access Journals (Sweden)

    Heath A Elrod

    Full Text Available Death receptor 5 (DR5 and caspase-8 are major components in the extrinsic apoptotic pathway. The alterations of the expression of these proteins during the metastasis of head and neck squamous cell carcinoma (HNSCC and their prognostic impact have not been reported. The present study analyzes the expression of DR5 and caspase-8 by immunohistochemistry (IHC in primary and metastatic HNSCCs and their impact on patient survival. Tumor samples in this study included 100 primary HNSCC with no evidence of metastasis, 100 primary HNSCC with lymph node metastasis (LNM and 100 matching LNM. IHC analysis revealed a significant loss or downregulation of DR5 expression in primary tumors with metastasis and their matching LNM compared to primary tumors with no evidence of metastasis. A similar trend was observed in caspase-8 expression although it was not statistically significant. Downregulation of caspase-8 and DR5 expression was significantly correlated with poorly differentiated tumors compared to moderately and well differentiated tumors. Univariate analysis indicates that, in HNSCC with no metastasis, higher expression of caspase-8 significantly correlated with better disease-free survival and overall survival. However, in HNSCC with LNM, higher caspase-8 expression significantly correlated with poorer disease-free survival and overall survival. Similar results were also generated when we combined both DR5 and caspase-8. Taken together, we suggest that both DR5 and caspase-8 are involved in regulation of HNSCC metastasis. Our findings warrant further investigation on the dual role of caspase-8 in cancer development.

  7. Nerve cells apoptosis and Fas and Caspase-8 expression among chronic alcoholic intoxication rats%慢性酒精中毒大鼠神经细胞凋亡及Fas、Caspase8蛋白的表达

    Institute of Scientific and Technical Information of China (English)

    段玉香; 张永利; 王彦

    2012-01-01

    目的 研究慢性酒精中毒大鼠神经细胞凋亡及Fas、Caspase8蛋白表达情况.方法 逐步增加饮水中酒精浓度,建立慢性酒精中毒大鼠动物模型,HE染色光镜下观察大鼠脑组织病理变化,Tunnel法检测大鼠脑细胞凋亡情况,免疫组化法检测Fas、Caspase8蛋白表达情况.结果 酒精组大脑皮质、海马、小脑等部位均有神经细胞数目缺失及细胞变性和损伤,酒精组各脑区凋亡细胞数和Fas、Casepse8蛋白表达阳性细胞数均高于对照组,两组比较差异有统计学意义(P<0.05).结论 慢性酒精中毒大鼠脑存在明显神经细胞凋亡,伴有Fas、Caspase-8蛋白表达增加,提示细胞死亡受体途径在慢性酒精中毒大鼠脑损伤病理过程中发挥作用.%Objective To understand nerve cells apoptosis and Fas and Casepse -8 protein expression status a-mong chronic alcoholic intoxication rats. Method Established chronic alcoholic intoxication rat model by increasing alcohol percentage of drinking gradually. Pathological changes were observed by light microscope, cells apoptosis and Fas and Casepse -8 protein expression were examined by Tunnel and Tmmunohistochemical method. Results In alcohol group, there were nerve cells missing, degeneration and damage in cerebral cortex, seahorse and cerebellum. The cells number of apoptosis and the positive cell of expression Fas and caspase -8 were obviously higher than control group, there was statistical significance between the 2 groups (P < 0.01) . Condusions There were obviously nerve cells apoptosis as well as the expression of Fas and Casepse -8 increased in the rats, which indicated that Fas and Casepse -8 protein attribute to nerve apoptosis and involved in the pathogenesis of chronic alcoholism.

  8. Cleavage by Caspase 8 and Mitochondrial Membrane Association Activate the BH3-only Protein Bid during TRAIL-induced Apoptosis.

    Science.gov (United States)

    Huang, Kai; Zhang, Jingjing; O'Neill, Katelyn L; Gurumurthy, Channabasavaiah B; Quadros, Rolen M; Tu, Yaping; Luo, Xu

    2016-05-27

    The BH3-only protein Bid is known as a critical mediator of the mitochondrial pathway of apoptosis following death receptor activation. However, since full-length Bid possesses potent apoptotic activity, the role of a caspase-mediated Bid cleavage is not established in vivo In addition, due to the fact that multiple caspases cleave Bid at the same site in vitro, the identity of the Bid-cleaving caspase during death receptor signaling remains uncertain. Moreover, as Bid maintains its overall structure following its cleavage by caspase 8, it remains unclear how Bid is activated upon cleavage. Here, Bid-deficient (Bid KO) colon cancer cells were generated by gene editing, and were reconstituted with wild-type or mutants of Bid. While the loss of Bid blocked apoptosis following treatment by TNF-related apoptosis inducing ligand (TRAIL), this blockade was relieved by re-introduction of the wild-type Bid. In contrast, the caspase-resistant mutant Bid(D60E) and a BH3 defective mutant Bid(G94E) failed to restore TRAIL-induced apoptosis. By generating Bid/Bax/Bak-deficient (TKO) cells, we demonstrated that Bid is primarily cleaved by caspase 8, not by effector caspases, to give rise to truncated Bid (tBid) upon TRAIL treatment. Importantly, despite the presence of an intact BH3 domain, a tBid mutant lacking the mitochondrial targeting helices (α6 and α7) showed diminished apoptotic activity. Together, these results for the first time establish that cleavage by caspase 8 and the subsequent association with the outer mitochondrial membrane are two critical events that activate Bid during death receptor-mediated apoptosis. PMID:27053107

  9. Caspase-8在不同浓度葡萄糖小鼠囊胚中的表达及意义

    Institute of Scientific and Technical Information of China (English)

    张丽萍; 李斌

    2011-01-01

    Objective To investigate the expression and significance of caspase - 8 in different concentrations of glucose in mouse blastocysts. Methods By superovulation, mouse 3. 5d blastocysts were obtained, expanded blastocysts from different females were pooled and randomly selected for one control and two experiments group,which exposed to doses of 0μmol/1, 7.5μmol/l, and 28. 0μmol/l glucose for 24h. Expression of Caspase -8 were detected by immunohistochemical S - P staining. The average optical density and the rate of positive area of expression of Caspase - 8 were analyzed using image analysis HPIAS - 2000 Analysis System. Results 1. The expression of Caspase -8:The expression of Caspase -8 was weakly positive and there was few light brown substance in cytoplasm of control mouse blastocysts. The expression of Caspase - 8 was negative in low glucose - treated mouse blastocysts, which cytoplasm was lack of staining. Deep staining of cytoplasm was found in high glucose - treated mouse blastocysts, where cells were obviously decreased. Conclusions High glucose also can up - regulate the expression of caspase - 8, and result in increased cell death in blastocysts, which will adversely affect the development and implantation of blastocysts.%目的 探讨Caspase-8在不同浓度葡萄糖小鼠囊胚中的表达及意义.方法 通过促超排卵,获取妊娠3.5d小鼠囊胚,随机分成三组,即对照组(空白)、低糖组(葡萄糖浓度为7.5mmol/L)和高糖组(葡萄糖浓度为28.0mmol/L),分别培养在含7.5mmol/L和28.0mmol/L葡萄糖的M199培养基中,培养24h后,然后再吸出囊胚.每组随机吸取30个囊胚用免疫组织化学S-P法,检测不同浓度葡萄糖对小鼠囊胚Caspase-8表达状况,利用HPIAS-1000图像分析系统测定Caspase-8在以上三组中表达的平均光密度和平均阳性面积率.结果 Caspase-8表达结果:空白组中囊胚细胞胞浆中可见少量浅棕黄色颗粒,Caspase-8表达呈弱阳性.低糖组中囊胚细胞胞浆未见着色,Caspase

  10. Neuronal deletion of caspase 8 protects against brain injury in mouse models of controlled cortical impact and kainic acid-induced excitotoxicity.

    Directory of Open Access Journals (Sweden)

    Maryla Krajewska

    Full Text Available Acute brain injury is an important health problem. Given the critical position of caspase 8 at the crossroads of cell death pathways, we generated a new viable mouse line (Ncasp8(-/-, in which the gene encoding caspase 8 was selectively deleted in neurons by cre-lox system.Caspase 8 deletion reduced rates of neuronal cell death in primary neuronal cultures and in whole brain organotypic coronal slice cultures prepared from 4 and 8 month old mice and cultivated up to 14 days in vitro. Treatments of cultures with recombinant murine TNFα (100 ng/ml or TRAIL (250 ng/mL plus cyclohexamide significantly protected neurons against cell death induced by these apoptosis-inducing ligands. A protective role of caspase 8 deletion in vivo was also demonstrated using a controlled cortical impact (CCI model of traumatic brain injury (TBI and seizure-induced brain injury caused by kainic acid (KA. Morphometric analyses were performed using digital imaging in conjunction with image analysis algorithms. By employing virtual images of hundreds of brain sections, we were able to perform quantitative morphometry of histological and immunohistochemical staining data in an unbiased manner. In the TBI model, homozygous deletion of caspase 8 resulted in reduced lesion volumes, improved post-injury motor performance, superior learning and memory retention, decreased apoptosis, diminished proteolytic processing of caspases and caspase substrates, and less neuronal degeneration, compared to wild type, homozygous cre, and caspase 8-floxed control mice. In the KA model, Ncasp8(-/- mice demonstrated superior survival, reduced seizure severity, less apoptosis, and reduced caspase 3 processing. Uninjured aged knockout mice showed improved learning and memory, implicating a possible role for caspase 8 in cognitive decline with aging.Neuron-specific deletion of caspase 8 reduces brain damage and improves post-traumatic functional outcomes, suggesting an important role for this

  11. ExoS of Pseudomonas aeruginosa induces apoptosis through a Fas receptor/caspase 8-independent pathway in HeLa cells.

    Science.gov (United States)

    Alaoui-El-Azher, Mounia; Jia, Jinghua; Lian, Wei; Jin, Shouguang

    2006-02-01

    Pseudomonas aeruginosa infection is a serious complication in immunocompromised individuals and in patients with cystic fibrosis. We have previously shown that the type III secreted effector ExoS triggers apoptosis in various cultured cell lines via its ADP-ribosyltransferase (ADPRT) activity. The apoptosis process was further shown to involve intrinsic signalling pathway requiring c-Jun N-terminal kinase (JNK)-initiated mitochondrial pathway. In the present study, we investigated the role of Fas pathway activation in P. aeruginosa-induced apoptosis. P. aeruginosa infection resulted in caspase 8 cleavage in HeLa cells, which was inhibited by overexpression of a dominant negative version of Fas-associated death domain (FADD), suggesting that Fas pathway was activated. In fact, confocal laser scanning microscopy showed that P. aeruginosa induced clustering of FasR. In addition, the ADPRT activity of the ExoS was required for the induction of FasR clustering and caspase 8 cleavage. However, blocking the FasR-FasL interaction by antagonistic antibodies to FasR or to FasL had no effect on P. aeruginosa-induced caspase 8 and caspase 3 activation, neither did the silencing of FasR by small interfering RNA (siRNA), suggesting that caspase 8 activation through the FADD bypasses FasR/FasL-mediated signalling. Thus, FADD-mediated caspase 8 activation involves intracellular ExoS in an ADPRT-dependent manner. Furthermore, silencing of caspase 8 by siRNA did not interfere with P. aeruginosa-induced apoptosis, whereas it rendered HeLa cells markedly increased resistance towards FasL-induced apoptosis. In conclusion, our findings indicate that ExoS of P. aeruginosa induces apoptosis through a mechanism that is independent of Fas receptor/caspase 8 pathway. PMID:16441442

  12. Staphylococcus aureus - induced tumor necrosis factor - related apoptosis - inducing ligand expression mediates apoptosis and caspase-8 activation in infected osteoblasts

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    Bost Kenneth L

    2003-04-01

    Full Text Available Abstract Background Staphylococcus aureus infection of normal osteoblasts induces expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL. Results Normal osteoblasts were incubated in the presence of purified bacterial products over a range of concentrations. Results demonstrate that purified surface structures and a selected superantigen present in the extracellular environment are not capable of inducing TRAIL expression by osteoblasts. Osteoblasts were co-cultured with S. aureus at various multiplicities of infection utilizing cell culture chamber inserts. Results of those experiments suggest that direct contact between bacteria and osteoblasts is necessary for optimal TRAIL induction. Finally, S. aureus infection of osteoblasts in the presence of anti-TRAIL antibody demonstrates that TRAIL mediates caspase-8 activation and apoptosis of infected cells. Conclusions Collectively, these findings suggest a mechanism whereby S. aureus mediates bone destruction via induction of osteoblast apoptosis.

  13. Compound K, a metabolite of ginseng saponin, induces apoptosis via caspase-8-dependent pathway in HL-60 human leukemia cells

    International Nuclear Information System (INIS)

    Compound K [20-O-β-(D-glucopyranosyl)-20(S)-protopanaxadiol], a metabolite of the protopanaxadiol-type saponins of Panax ginseng C.A. Meyer, has been reported to possess anti-tumor properties to inhibit angiogenesis and to induce tumor apoptosis. In the present study, we investigated the effect of Compound K on apoptosis and explored the underlying mechanisms involved in HL-60 human leukemia cells. We examined the effect of Compound K on the viabilities of various cancer cell lines using MTT assays. DAPI assay, Annexin V and PI double staining, Western blot assay and immunoprecipitation were used to determine the effect of Compound K on the induction of apoptosis. Compound K was found to inhibit the viability of HL-60 cells in a dose- and time-dependent manner with an IC50 of 14 μM. Moreover, this cell death had typical features of apoptosis, that is, DNA fragmentation, DNA ladder formation, and the externalization of Annexin V targeted phosphatidylserine residues in HL-60 cells. In addition, compound-K induced a series of intracellular events associated with both the mitochondrial- and death receptor-dependent apoptotic pathways, namely, (1) the activation of caspases-3, -8, and -9; (2) the loss of mitochondrial membrane potential; (3) the release of cytochrome c and Smac/DIABLO to the cytosol; (4) the translocation of Bid and Bax to mitochondria; and (5) the downregulations of Bcl-2 and Bcl-xL. Furthermore, a caspase-8 inhibitor completely abolished caspase-3 activation, Bid cleavage, and subsequent DNA fragmentation by Compound K. Interestingly, the activation of caspase-3 and -8 and DNA fragmentation were significantly prevented in the presence of cycloheximide, suggesting that Compound K-induced apoptosis is dependent on de novo protein synthesis. The results indicate that caspase-8 plays a key role in Compound K-stimulated apoptosis via the activation of caspase-3 directly or indirectly through Bid cleavage, cytochrome c release, and caspase-9 activation

  14. Dietary n-3 PUFAs augment caspase 8 activation in Staphylococcal aureus enterotoxin B stimulated T-cells.

    Science.gov (United States)

    Gill, R; Jen, K L; McCabe, M J J; Rosenspire, A

    2016-10-15

    Epidemiological studies have linked consumption of n-3 PUFAs with a variety of beneficial health benefits, particularly with respect to putative anti-inflammatory effects. Unfortunately, many of these results remain somewhat controversial because in most instances there has not been a linkage to specific molecular mechanisms. For instance, dietary exposure to low levels of mercury has been shown to be damaging to neural development, but concomitant ingestion of n-3 PUFAs as occurs during consumption of fish, has been shown to counteract the detrimental effects. As the mechanisms mediating the neurotoxicity of environmental mercury are not fully delineated, it is difficult to conceptualize a testable molecular mechanism explaining how n-3 PUFAs negate its neurotoxic effects. However, environmental exposure to mercury also has been linked to increased autoimmunity. By way of a molecular understanding of this immuno-toxic association, disruption of CD95 signaling is well established as a triggering factor for autoimmunity, and we have previously shown that environmentally relevant in vitro and dietary exposures to mercury interfere with CD95 signaling. In particular we have shown that activation of caspase 8, as well as downstream activation of caspase 3, in response to CD95 agonist stimulation is depressed by mercury. More recently we have shown in vitro that the n-3 PUFA docosahexaenoic acid counteracts the negative effect of mercury on CD95 signaling by restoring caspase activity. We hypothesized that concomitant ingestion of n-3 PUFAs with mercury might be protective from the immuno-toxic effects of mercury, as it is with mercury's neuro-toxic effects, and in the case of immuno-toxicity this would be related to restoration of CD95 signal strength. We now show that dietary ingestion of n-3 PUFAs generally promotes CD95 signaling by upregulating caspase 8 activation. Apart from accounting for the ability of n-3 PUFAs to specifically counteract autoimmune sequelae of

  15. Compound K, a metabolite of ginseng saponin, induces apoptosis via caspase-8-dependent pathway in HL-60 human leukemia cells

    Directory of Open Access Journals (Sweden)

    Choi Jung-Hye

    2009-12-01

    Full Text Available Abstract Background Compound K [20-O-β-(D-glucopyranosyl-20(S-protopanaxadiol], a metabolite of the protopanaxadiol-type saponins of Panax ginseng C.A. Meyer, has been reported to possess anti-tumor properties to inhibit angiogenesis and to induce tumor apoptosis. In the present study, we investigated the effect of Compound K on apoptosis and explored the underlying mechanisms involved in HL-60 human leukemia cells. Methods We examined the effect of Compound K on the viabilities of various cancer cell lines using MTT assays. DAPI assay, Annexin V and PI double staining, Western blot assay and immunoprecipitation were used to determine the effect of Compound K on the induction of apoptosis. Results Compound K was found to inhibit the viability of HL-60 cells in a dose- and time-dependent manner with an IC50 of 14 μM. Moreover, this cell death had typical features of apoptosis, that is, DNA fragmentation, DNA ladder formation, and the externalization of Annexin V targeted phosphatidylserine residues in HL-60 cells. In addition, compound-K induced a series of intracellular events associated with both the mitochondrial- and death receptor-dependent apoptotic pathways, namely, (1 the activation of caspases-3, -8, and -9; (2 the loss of mitochondrial membrane potential; (3 the release of cytochrome c and Smac/DIABLO to the cytosol; (4 the translocation of Bid and Bax to mitochondria; and (5 the downregulations of Bcl-2 and Bcl-xL. Furthermore, a caspase-8 inhibitor completely abolished caspase-3 activation, Bid cleavage, and subsequent DNA fragmentation by Compound K. Interestingly, the activation of caspase-3 and -8 and DNA fragmentation were significantly prevented in the presence of cycloheximide, suggesting that Compound K-induced apoptosis is dependent on de novo protein synthesis. Conclusions The results indicate that caspase-8 plays a key role in Compound K-stimulated apoptosis via the activation of caspase-3 directly or indirectly through

  16. Caspase-8 Deficiency Presenting as Late-Onset Multi-Organ Lymphocytic Infiltration with Granulomas in two Adult Siblings.

    Science.gov (United States)

    Niemela, Julie; Kuehn, Hye Sun; Kelly, Corin; Zhang, Mingchang; Davies, Joie; Melendez, Jose; Dreiling, Jennifer; Kleiner, David; Calvo, Katherine; Oliveira, João B; Rosenzweig, Sergio D

    2015-05-01

    Caspase-8 deficiency (CED) was originally described in 2002 in two pediatric patients presenting with clinical manifestations resembling autoimmune lymphoproliferative syndrome (ALPS) accompanied by infections, and T, B and NK cell defects. Since then, no new CED patients were published. Here we report two adult siblings (Pt1 and Pt2) presenting in their late thirties with pulmonary hypertension leading to lung transplant (Pt1), and a complex neurological disease leading to multiple cranial nerves palsies (Pt2) as their main manifestations. A thorough clinical and immunological evaluation was performed at the Primary Immunodeficiency Clinic at NIH, followed by whole exome sequencing. The patients had multiorgan lymphocytic infiltration and granulomas, as well as clinical signs of immune deficiency/ immune dysregulation. Both siblings carried homozygous mutations in CASP8, c.1096C > T, p.248R > W. This was the same mutation described on the previously published CED patients, to whom these new patients were likely distantly related. We report two new CED patients presenting during adulthood with life-threatening end-organ lymphocyte infiltrates affecting the lungs, liver, spleen, bone marrow and central nervous system. This phenotype broadens the clinical spectrum of manifestations associated with this disease and warrants the search of CASP8 mutations in other cohorts of patients. PMID:25814141

  17. A ginseng saponin metabolite-induced apoptosis in HepG2 cells involves a mitochondria-mediated pathway and its downstream caspase-8 activation and Bid cleavage

    International Nuclear Information System (INIS)

    20-O-(β-D-Glucopyranosyl)-20(S)-protopanaxadiol (IH901), an intestinal bacterial metabolite of ginseng saponin formed from ginsenosides Rb1, Rb2, and Rc, is suggested to be a potential chemopreventive agent. Here, we show that IH901 induces apoptosis in human hepatoblastoma HepG2 cells. IH901 led to an early activation of procaspase-3 (12 h posttreatment), and the activation of caspase-8 became evident only later (18 h posttreatment). Caspase activation was a necessary requirement for apoptosis because caspase inhibitors significantly inhibited cell death by IH901. Treatment of HepG2 cells with IH901 also induced the cleavage of cytosolic factors such as Bid and Bax and translocation of truncated Bid (tBid) to mitochondria. A time-dependent release of cytochrome c from mitochondria was observed, which was accompanied by activation of caspase-9. A broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), and a specific inhibitor for caspase-8, N-benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone (zIETD-fmk), abrogated Bid processing and translocation, and caspase-3 activation. Cytochrome c release was inhibited by zVAD-fmk, however, the inhibition by zIETD-fmk was not complete. The activation of caspase-8 was inhibited not only by zIETD-fmk but also by zVAD-fmk. The results, together with the kinetic change of caspase activation, indicate that activation of caspase-8 occurred downstream of caspase-3 and -9. Our data suggest that the activation of caspase-8 after early caspase-3 activation might act as an amplification loop necessary for successful apoptosis. Primary hepatocytes isolated from normal Sprague-Dawley rats were not affected by IH901 (0-60 μM). The very low toxicity in normal hepatocytes and high activity in hepatoblastoma HepG2 cells suggest that IH901 is a promising experimental cancer chemopreventive agent

  18. A delay prior to mitotic entry triggers caspase 8-dependent cell death in p53-deficient Hela and HCT-116 cells.

    Science.gov (United States)

    Silva, Victoria C; Plooster, Melissa; Leung, Jessica C; Cassimeris, Lynne

    2015-01-01

    Stathmin/Oncoprotein 18, a microtubule destabilizing protein, is required for survival of p53-deficient cells. Stathmin-depleted cells are slower to enter mitosis, but whether delayed mitotic entry triggers cell death or whether stathmin has a separate pro-survival function was unknown. To test these possibilities, we abrogated the cell cycle delay by inhibiting Wee1 in synchronized, stathmin-depleted cells and found that apoptosis was reduced to control levels. Synchronized cells treated with a 4 hour pulse of inhibitors to CDK1 or both Aurora A and PLK1 delayed mitotic entry and apoptosis was triggered only in p53-deficient cells. We did not detect mitotic defects downstream of the delayed mitotic entry, indicating that cell death is activated by a mechanism distinct from those activated by prolonged mitotic arrest. Cell death is triggered by initiator caspase 8, based on its cleavage to the active form and by rescue of viability after caspase 8 depletion or treatment with a caspase 8 inhibitor. In contrast, initiator caspase 9, activated by prolonged mitotic arrest, is not activated and is not required for apoptosis under our experimental conditions. P53 upregulates expression of cFLIPL, a protein that blocks caspase 8 activation. cFLIPL levels are lower in cells lacking p53 and these levels are reduced to a greater extent after stathmin depletion. Expression of FLAG-tagged cFLIPL in p53-deficient cells rescues them from apoptosis triggered by stathmin depletion or CDK1 inhibition during G2. These data indicate that a cell cycle delay in G2 activates caspase 8 to initiate apoptosis specifically in p53-deficient cells.

  19. Influence of 5-Azacytidine on Drug Resistance of Caspase-8 Silence Neuroblastoma%去甲基药5-氮杂胞苷对Caspase-8缺乏的神经母细胞瘤细胞化疗敏感性的影响

    Institute of Scientific and Technical Information of China (English)

    周广玉; 李爱敏; 初清; 王晓莉

    2010-01-01

    目的 探讨去甲基5-氮杂胞苷能否恢复神经母细胞瘤(NB)细胞中Caspase-8的表达水平,从而提高NB细胞SY5Y对常用化疗药物依托泊苷的敏感性.方法 采用反转录-PCR(RT-PCR)技术检测5-氮杂胞苷作用前后Caspase-8 mRNA的表达.Elivision二步法检测5-氮杂胞苷作用前后SY5Y细胞中Caspase-8蛋白的表达.应用四甲基偶氮唑蓝(MTT)法检测Caspase-8缺乏的NB细胞中加入化疗依托泊苷后细胞存活率,比较加入5-氮杂咆苷后再入依托泊苷后细胞存活率的变化.结果 RT-PCR方法未检测到SY5Y细胞中Caspase-8 mRNA表达,5-氮杂胞苷单独作用3d可以检测到Caspase-8 mRNA表达,5d组Caspase-8 mRNA表达较3d组明显增强,且Caspase-8 mRNA表达水平随5-氮杂胞苷水平增高而增高.Elivision二步法检测SY5Y细胞中Caspase-8蛋白表达阴性,5-氮杂胞苷作用3d后Caspase-8蛋白表达呈阳性.MTT法检测发现小同质量浓度的依托泊苷(0.1、0.3、1.0、2.5)mg·L-1单独作用24h组细胞存活率分别为94.16%±5.89%、76.46%±5.83%、44.31%±5 05%、36.15%±5.71%;5-氮杂胞苷+同质量浓度依托泊苷组细胞存活率分别为84.23%±6 95%,61.00%±4.98%,31.64%±3.84%,23.75%±1.89%,联合作用组细胞存活率较单独作用组细胞存活率明显下降,其差异均有统计学意义(Pa<0.01).结论 SY5Y细胞不表达Caspase-8 mRNA及蛋白,5-氮杂胞苷可诱导SY5Y细胞Caspase-8 mRNA及蛋白表达,且随时间延长,Caspase-8 mRNA表达随之增加.依托泊苷对NB细胞SY5Y具有增殖抑制作用.5-氮杂胞苷可增强其对SY5Y的增殖抑制作用,并提高化疗的敏感性.

  20. Unusual roles of caspase-8 in triple-negative breast cancer cell line MDA-MB-231.

    Science.gov (United States)

    De Blasio, Anna; Di Fiore, Riccardo; Morreale, Marco; Carlisi, Daniela; Drago-Ferrante, Rosa; Montalbano, Mauro; Scerri, Christian; Tesoriere, Giovanni; Vento, Renza

    2016-06-01

    Triple-negative breast cancer (TNBC) is a clinically aggressive form of breast cancer that is unresponsive to endocrine agents or trastuzumab. TNBC accounts for ~10-20% of all breast cancer cases and represents the form with the poorest prognosis. Patients with TNBC are at higher risk of early recurrence, mainly in the lungs, brain and soft tissue, therefore, there is an urgent need for new therapies. The present study was carried out in MDA-MB-231 cells, where we assessed the role of caspase-8 (casp-8), a critical effector of death receptors, also involved in non‑apoptotic functions. Analysis of casp-8 mRNA and protein levels indicated that they were up-regulated with respect to the normal human mammalian epithelial cells. We demonstrated that silencing of casp-8 by small interfering-RNA, strongly decreased MDA-MB-231 cell growth by delaying G0/G1- to S-phase transition and increasing p21, p27 and hypo-phosphorylated/active form of pRb levels. Surprisingly, casp-8-knockdown, also potently increased both the migratory and metastatic capacity of MDA-MB‑231 cells, as shown by both wound healing and Matrigel assay, and by the expression of a number of related-genes and/or proteins such as VEGFA, C-MYC, CTNNB1, HMGA2, CXCR4, KLF4, VERSICAN V1 and MMP2. Among these, KLF4, a transcriptional factor with a dual role (activator and repressor), seemed to play critical roles. We suggest that in MDA-MB‑231 cells, the endogenous expression of casp-8 might keep the cells perpetually cycling through downregulation of KLF4, the subsequent lowering of p21 and p27, and the inactivation by hyperphosphorylation of pRb. Simultaneously, by lowering the expression of some migratory and invasive genes, casp-8 might restrain the metastatic ability of the cells. Overall, our findings showed that, in MDA-MB-231 cells, casp-8 might play some unusual roles which should be better explored, in order to understand whether it might be identified as a molecular therapeutic target. PMID

  1. Harmol induces apoptosis by caspase-8 activation independently of Fas/Fas ligand interaction in human lung carcinoma H596 cells.

    Science.gov (United States)

    Abe, Akihisa; Yamada, Hiroyuki

    2009-06-01

    The beta-carboline alkaloids are naturally existing plant substances. It is known that these alkaloids have a wide spectrum of neuropharmacological, psychopharmacological, and antitumor effects. Therefore, they have been traditionally used in oriental medicine for the treatment of various diseases including cancers and malaria. In this study, harmol and harmalol, which are beta-carboline alkaloids, were examined for their antitumor effect on human lung carcinoma cell lines, and structure-activity relationship was also investigated. H596, H226, and A549 cells were treated with harmol and harmalol, respectively. Apoptosis was induced by harmol only in H596 cells. In contrast, harmalol had negligible cytotoxicity in three cell lines. Harmol induced caspase-3, caspase-8, and caspase-9 activities and caspase-3 activities accompanied by cleavage of poly-(ADP-ribose)-polymerase. Furthermore, harmol treatment decreased the native Bid protein, and induced the release of cytochrome c from mitochondria to cytosol. The apoptosis induced by harmol was completely inhibited by caspase-8 inhibitor and partially inhibited by caspase-9 inhibitor. The antagonistic antibody ZB4 blocked Fas ligand-induced apoptosis, but had no effect on harmol-induced apoptosis. Harmol had no significant effect on the expression of Fas. In conclusion, our results showed that the harmol could cause apoptosis-inducing effects in human lung H596 cells through caspase-8-dependent pathway but independent of Fas/Fas ligand interaction. PMID:19318910

  2. pERK 1/2 inhibit Caspase-8 induced apoptosis in cancer cells by phosphorylating it in a cell cycle specific manner.

    Science.gov (United States)

    Mandal, Ranadip; Raab, Monika; Matthess, Yves; Becker, Sven; Knecht, Rainald; Strebhardt, Klaus

    2014-03-01

    ERK 1/2 are found to be hyperactive in many cancers. Active ERK 1/2 (pERK 1/2) are known to protect cancer cells from undergoing death receptor-mediated apoptosis, although the mechanism(s) behind this is poorly understood. Through in vitro kinase assays and mass-spectrometry we demonstrate that pERK 1/2 can phosphorylate pro-Caspase-8 at S387. Also, in EGFR-overexpressing Type I and II ovarian and breast cancer cell lines respectively, ERK 1/2 remain active only during the interphase. During this period, pERK 1/2 could inhibit Trail-induced apoptosis, most effectively during the G1/S phase. By knocking-down the endogenous pro-Caspase-8 using RNAi and replacing it with its non-phosphorylatable counterpart (S387A), a significant increase in Caspase-8 activity upon Trail stimulation was observed, even in the presence of pERK 1/2. Taken together, we propose that a combination of Trail and an inhibitor of ERK 1/2 activities could potentially enhance of Trail's effectiveness as an anti-cancer agent in ERK 1/2 hyperactive cancer cells.

  3. Docosahexaenoic acid induces apoptosis in MCF-7 cells in vitro and in vivo via reactive oxygen species formation and caspase 8 activation.

    Directory of Open Access Journals (Sweden)

    Ki Sung Kang

    Full Text Available BACKGROUND: The present study sought to further investigate the in vitro and in vivo anticancer effects of a representative omega-3 fatty acid, docosahexaenoic acid (DHA, with a focus on assessing the induction of oxidative stress and apoptosis as an important mechanism for its anticancer actions. METHODOLOGY/PRINCIPAL FINDINGS: In vitro studies showed that DHA strongly reduces the viability and DNA synthesis of MCF-7 human breast cancer cells in culture, and also promotes cell death via apoptosis. Mechanistically, accumulation of reactive oxygen species and activation of caspase 8 contribute critically to the induction of apoptotic cell death. Co-presence of antioxidants or selective inhibition or knockdown of caspase 8 each effectively abrogates the cytotoxic effect of DHA. Using athymic nude mice as an in vivo model, we found that feeding animals the 5% fish oil-supplemented diet for 6 weeks significantly reduces the growth of MCF-7 human breast cancer cells in vivo through inhibition of cancer cell proliferation as well as promotion of cell death. Using 3-nitrotyrosine as a parameter, we confirmed that the fish oil-supplemented diet significantly increases oxidative stress in tumor cells in vivo. Analysis of fatty acid content in plasma and tissues showed that feeding animals a 5% fish oil diet increases the levels of DHA and eicosapentaenoic acid in both normal and tumorous mammary tissues by 329% and 300%, respectively. CONCLUSIONS/SIGNIFICANCE: DHA can strongly induce apoptosis in human MCF-7 breast cancer cells both in vitro and in vivo. The induction of apoptosis in these cells is selectively mediated via caspase 8 activation. These observations call for further studies to assess the effectiveness of fish oil as a dietary supplement in the prevention and treatment of human breast cancer.

  4. Deletion of caspase-8 in mouse myeloid cells blocks microglia pro-inflammatory activation and confers protection in MPTP neurodegeneration model.

    Science.gov (United States)

    Kavanagh, Edel; Burguillos, Miguel Angel; Carrillo-Jimenez, Alejandro; Oliva-Martin, María José; Santiago, Martiniano; Rodhe, Johanna; Joseph, Bertrand; Venero, Jose Luis

    2015-09-01

    Increasing evidence involves sustained pro-inflammatory microglia activation in the pathogenesis of different neurodegenerative diseases, particularly Parkinson's disease (PD). We recently uncovered a completely novel and unexpected role for caspase-8 and its downstream substrates caspase-3/7 in the control of microglia activation and associated neurotoxicity to dopaminergic cells. To demonstrate the genetic evidence, mice bearing a floxed allele ofCASP8 were crossed onto a transgenic line expressing Cre under the control of Lysozyme 2 gene. Analysis of caspase-8 gene deletion in brain microglia demonstrated a high efficiency in activated but not in resident microglia. Mice were challenged with lipopolysaccharide, a potent inducer of microglia activation, or with MPTP, which promotes specific dopaminergic cell damage and consequent reactive microgliosis. In neither of these models, CASP8 deletion appeared to affect the overall number of microglia expressing the pan specific microglia marker, Iba1. In contrast, CD16/CD32 expression, a microglial pro-inflammatory marker, was found to be negatively affected upon CASP8 deletion. Expression of additional proinflammatory markers were also found to be reduced in response to lipopolysaccharide. Of importance, reduced pro-inflammatory microglia activation was accompanied by a significant protection of the nigro-striatal dopaminergic system in the MPTP mouse model of PD.

  5. TNF-alpha-induced mitochondrial alterations in human T cells requires FADD and caspase-8 activation but not RIP and caspase-3 activation.

    Science.gov (United States)

    Shakibaei, Mehdi; Sung, Bokyung; Sethi, Gautam; Aggarwal, Bharat B

    2010-09-15

    Although much is known about how TNF-alpha induces apoptosis in the presence of inhibitors of protein synthesis, little is known about how it induces apoptosis without these inhibitors. In this report we investigated temporal sequence of events induced by TNF-alpha in the absence of protein synthesis. Regardless of whether we measured the effects by plasma membrane phosphotidylserine accumulation, by DNA strand breaks, or activation of caspases, significant changes were observed only between 12-24 h of TNF-alpha treatment. One of the earliest changes observed after TNF-alpha treatment was mitochondrial swelling at 10 min; followed by cytochrome c and Smac release at 10-30 min, and then heterochromatin clumping occurred at 60 min. While genetic deletion of receptor-interaction protein (RIP) had no effect on TNF-alpha-induced mitochondrial damage, deletion of Fas-associated death domain (FADD) abolished the TNF-induced mitochondrial swelling. Since pan-caspase inhibitor z-VAD-fmk abolished the TNF-alpha-induced mitochondrial changes, z-DEVD-fmk, an inhibitor of caspase-3 had no effect, suggesting that TNF-alpha-induced mitochondrial changes or cytochrome c and Smac release requires caspase-8 but not caspase-3 activation. Overall, our results indicated that mitochondrial changes are early events in TNF-alpha-induced apoptosis and that these mitochondrial changes require recruitment of FADD and caspase-8 activation, but not caspase-3 activation or RIP recruitment. PMID:20136500

  6. Mycobacterium tuberculosis-infected human monocytes down-regulate microglial MMP-2 secretion in CNS tuberculosis via TNFα, NFκB, p38 and caspase 8 dependent pathways

    Directory of Open Access Journals (Sweden)

    Elkington Paul T

    2011-05-01

    Full Text Available Abstract Tuberculosis (TB of the central nervous system (CNS is a deadly disease characterized by extensive tissue destruction, driven by molecules such as Matrix Metalloproteinase-2 (MMP-2 which targets CNS-specific substrates. In a simplified cellular model of CNS TB, we demonstrated that conditioned medium from Mycobacterium tuberculosis-infected primary human monocytes (CoMTb, but not direct infection, unexpectedly down-regulates constitutive microglial MMP-2 gene expression and secretion by 72.8% at 24 hours, sustained up to 96 hours (P M.tb-infected monocyte-dependent networks paradoxically involves the pro-inflammatory mediators TNF-α, p38 MAP kinase and NFκB in addition to a novel caspase 8-dependent pathway.

  7. Autoregulatory Feedback Mechanism of P38MAPK/Caspase-8 in Photodynamic Therapy-Hydrophilic/Lipophilic Tetra-α-(4-carboxyphenoxy Phthalocyanine Zinc-Induced Apoptosis of Human Hepatocellular Carcinoma Bel-7402 Cells

    Directory of Open Access Journals (Sweden)

    Yu Wang

    2014-01-01

    Full Text Available Photodynamic therapy (PDT is a novel and promising antitumor treatment. Our previous study showed that hydrophilic/lipophilic tetra-α-(4-carboxyphenoxy phthalocyanine zinc- (TαPcZn- mediated PDT (TαPcZn-PDT inhibits the proliferation of human hepatocellular carcinoma Bel-7402 cells by triggering apoptosis and arresting cell cycle. However, mechanisms of TαPcZn-PDT-induced apoptosis of Bel-7402 cells have not been fully clarified. In the present study, therefore, effect of TαPcZn-PDT on apoptosis, P38MAPK, p-P38MAPK, Caspase-8, Caspase-3, Bcl-2, Bid, Cytochrome c, and mitochondria membrane potential in Bel-7402 cells without or with P38MAPK inhibitor SB203580 or Caspase-8 inhibitor Ac-IEFD-CHO was investigated by haematoxylin and eosin (HE staining assay, flow cytometry analysis of annexin V-FITC/propidium iodide (PI double staining cells and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1, and immunoblot assay. We found that TαPcZn-PDT resulted in apoptosis induction, activation of P38MAPK, Caspase-8, Caspase-3, and Bid, downregulation of Bcl-2, release of Cytochrome c from mitochondria, and disruption of mitochondrial membrane potential in TαPcZn-PDT-treated Bel-7402 cells. In contrast, SB203580 or Ac-IEFD-CHO attenuated induction of apoptosis, activation of P38MAPK, Caspase-8, Caspase-3, and Bid, downregulation of Bcl-2, release of Cytochrome c from mitochondria, and disruption of mitochondrial membrane potential in TαPcZn-PDT-treated Bel-7402 cells. Taken together, we conclude that Caspase-3, Bcl-2, Bid, and mitochondria are involved in autoregulatory feedback of P38MAPK/Caspase-8 during TαPcZn-PDT-induced apoptosis of Bel-7402 cells.

  8. Memantine prevents cognitive impairment and reduces Bcl-2 and caspase 8 immunoreactivity in rats injected with amyloid β1-40.

    Science.gov (United States)

    Miguel-Hidalgo, José Javier; Paul, Ian A; Wanzo, Valerie; Banerjee, Pradeep K

    2012-10-01

    Amyloid-beta peptides (Aβ) can trigger apoptotic cascades in neurons. We found previously that memantine, an uncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptors approved for the treatment of moderate to severe Alzheimer's disease, can prevent neurodegeneration induced by intracranial Aβ(1-40) injection. In this study, we tested the hypothesis that memantine prevents Aβ(1-40)-mediated cognitive impairment, neurodegeneration, and apoptosis of hippocampal neurons in rats. In addition, we hypothesized that Aβ(1-40) injection would induce changes in the levels of one or more apoptosis-related proteins, and that these changes would be attenuated by memantine treatment. Female Sprague-Dawley rats were administered memantine (continuous subcutaneous application, 9.6-14.4mg/kg/day; n=8) or vehicle (water; n=8) for 9 days. Two days after treatment initiation, the animals were bilaterally injected with Aβ(1-40) into the CA1/DG region of the hippocampus, subjected to active avoidance testing for 7 days, and sacrificed for immunohistochemical examination of four caspases (3, 6, 8, and 9) and three proteins of the Bcl-2 family (Bcl-2, Bax, and Bad). Injection of Aβ resulted in neurodegeneration, DNA fragmentation, increased Bcl-2 immunostaining, and significantly impaired performance in an active avoidance task, all which were significantly attenuated in rats treated with memantine. No differences in immunoreactivity of caspases 3, 6, 8, and 9 were discovered between groups after 7 days. Additional experiments demonstrated that an increase in caspase 8 immunostaining, observed 3 days after Aβ(1-40) injection, was significantly attenuated in memantine-treated rats. These data suggest that, in rats, memantine can prevent amyloid-triggered expression of apoptosis-related markers and concomitant cognitive deficits. PMID:22824463

  9. Synergistic Effect of Subtoxic-dose Cisplatin and TRAIL to Mediate Apoptosis by Down-regulating Decoy Receptor 2 and Up-regulating Caspase-8, Caspase-9 and Bax Expression on NCI-H460 and A549 Cells

    Directory of Open Access Journals (Sweden)

    Xiaoyan Zhang

    2013-05-01

    Full Text Available Objective(s: Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL can selectively induce apoptosis in tumor cells, more than half of tumors including non-small cell lung cancer (NSCLC exhibit TRAIL-resistance. The purpose of this study was to determine whether subtoxic-dose cisplatin and TRAIL could synergistically enhance apoptosis on NSCLC cells and investigate its underlying mechanisms. Materials and Methods:NCI-H460 and A549 cells were treated with TRAIL alone, cisplatin alone or combination treatment in this study. The cytotoxicity was evaluated according to Sulforhodamine B assay, and apoptosis was examined using Hoechst 33342 staining and flow cytometry. The mRNA and protein levels of TRAIL receptors and apoptotic proteins including caspase-8, caspase-9, Bcl-2 and Bax were determined by RT-PCR and Western blotting, respectively. Results:Our results showed that NCI-H460 cells were sensitive to TRAIL, whereas A549 cells were resistant. However, subtoxic-dose cisplatin could enhance the both cells to TRAIL-mediated cell proliferation inhibition and apoptosis. The underlying mechanisms might be associated with the down-regulation of DcR2 and up-regulation of Caspase-8, Caspase-9 and Bax. Conclusion:Subtoxic-dose cisplatin could enhance both TRAIL- sensitive and TRAIL- resistant NSCLC cells to TRAIL-mediated apoptosis. These findings motivated further studies to evaluate such a combinatory therapeutic strategy against NSCLC in the animal models.

  10. Inclusion Complex of Zerumbone with Hydroxypropyl-β-Cyclodextrin Induces Apoptosis in Liver Hepatocellular HepG2 Cells via Caspase 8/BID Cleavage Switch and Modulating Bcl2/Bax Ratio

    Directory of Open Access Journals (Sweden)

    Nabilah Muhammad Nadzri

    2013-01-01

    Full Text Available Zerumbone (ZER isolated from Zingiber zerumbet was previously encapsulated with hydroxypropyl-β-cyclodextrin (HPβCD to enhance ZER’s solubility in water, thus making it highly tolerable in the human body. The anticancer effects of this new ZER-HPβCD inclusion complex via apoptosis cell death were assessed in this study for the first time in liver hepatocellular cells, HepG2. Apoptosis was ascertained by morphological study, nuclear stain, and sub-G1 cell population accumulation with G2/M arrest. Further investigations showed the release of cytochrome c and loss of mitochondrial membrane potential, proving mitochondrial dysfunction upon the ZER-HPβCD treatment as well as modulating proapoptotic and anti-apototic Bcl-2 family members. A significant increase in caspase 3/7, caspase 9, and caspase 8 was detected with the depletion of BID cleaved by caspase 8. Collectively, these results prove that a highly soluble inclusion complex of ZER-HPβCD could be a promising anticancer agent for the treatment of hepatocellular carcinoma in humans.

  11. TRAIL Activates a Caspase 9/7-Dependent Pathway in Caspase 8/10-Defective SK-N-SH Neuroblastoma Cells with Two Functional End Points: Induction of Apoptosis and PGE2 Release

    Directory of Open Access Journals (Sweden)

    Giorgio Zauli

    2003-09-01

    Full Text Available Most neuroblastoma cell lines do not express apical caspases 8 and 10, which play a key role in mediating tumor necrosis factor-related apoptosis-inducing ligand (TRAIL cytotoxicity in a variety of malignant cell types. In this study, we demonstrated that TRAIL induced a moderate but significant increase of apoptosis in the caspase 8/10-deficient SK-N-SH neuroblastoma cell line, through activation of a novel caspase 9/7 pathway. Concomitant to the induction of apoptosis, TRAIL also promoted a significant increase of prostaglandin E2 (PGE2 release by SKN-SH cells. Moreover, coadministration of TRAIL plus indomethacin, a pharmacological inhibitor of cyclooxygenase (COX, showed an additive effect on SKN-SH cell death. In spite of the ability of TRAIL to promote the phosphorylation of both ERKi/2 and p38/MAPK, which have been involved in the control of COX expression/activity, neither PD98059 nor SB203580, pharmacological inhibitors of the ERKi/2 and p38/MAPK pathways, respectively, affected either PGE2 production or apoptosis induced by TRAIL. Finally, both induction of apoptosis and PGE2 release were completely abrogated by the broad caspase inhibitor z-VAD4mk, suggesting that both biologic end points were regulated in SK-N-SH cells through a caspase 9/7-dependent pathway.

  12. 半胱氨酸蛋白酶-8和P53在慢性砷中毒大鼠肾近端小管表达的研究%Expression of cysteine caspase-8 and P53 in renal proximal tubular epithelial cell of chronic arsenic poisoning rats

    Institute of Scientific and Technical Information of China (English)

    钱立全; 李远慧; 孔祥照; 金婷婷; 李娜

    2012-01-01

    Objective To study the molecular mechanism of renal injury of chronic arsenic poisoning rats induced by the expression of cysteine caspase-8 and P53 in renal proximal tubular epithelial cells.Methods Sixty healthy SD rats were divided into three groups,high-,low-dose group,and control group,n =20 in each group.The rats in high and low dose groups were treated with As203 through drinking water,10.0 and 0.4 mg/kg,respectively.The control rats were given distilled water.Four months later,serum and urinary arsenic level was determined,and kidney specimens were taken.The expression of cysteine caspase-8 and P53 in renal proximal tubular epithelial cells was detected by histological technique-HE staining and SABC immunohistochemistry.In addition,cell number counting and image analyses were used in the study.Results The number of caspase-8 positive cells of renal proximal tubule in control group,low-and high-dose group was 3.33±1.32,31.14±8.02 and 46.50±7.20 cell number/visual fields,respectively,which was increased with dose increasing(all P <0.05);the average gray value was 151.34±6.40,133.58±4.63 and 128.34±16.28,respectively,decreased with dose increasing(all P <0.05).The number of P53 positive cells was 3.17±1.59,26.29±4.23 and 47.00±6.22 cell number/visual fields,respectively,increased with dose increasing (all P < 0.05) ; the average gray value was 142.54±8.06,121.48±5.68 and 101.89±6.35,respectively,decreased with dose increasing (all P < 0.05).Conclusion The increase of caspase-8 and P53 positive cells is one of the molecular mechanisms of renal injury induced by arsenic poisoning.%目的 探讨慢性砷中毒大鼠肾近端小管细胞半胱氨酸蛋白酶-8(caspase-8)和P53表达致肾脏损伤的分子机制.方法 60只SD大鼠分为对照组,低、高剂量组,每组20只,高、低剂量组分别给予三氧化二砷(As2O3) 10.0、0.4 mg/kg水溶液自由饮用,对照组自由饮用蒸馏水.分笼4个月,测定血砷、尿砷,取肾脏

  13. Inference of Isoforms from Short Sequence Reads

    Science.gov (United States)

    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.

  14. Isolation and characterization of patatin isoforms

    NARCIS (Netherlands)

    Pots, A.M.; Gruppen, H.; Hessing, M.; Boekel, M.A.J.S. van; Voragen, A.G.J.

    1999-01-01

    Patatin has, so far, been considered a homogeneous group of proteins. A comparison of the isoforms in terms of structural properties or stability has not been reported. A method to obtain various isoform fractions as well as a comparison of the physicochemical properties of these pools is presented.

  15. Functional studies of sodium pump isoforms

    DEFF Research Database (Denmark)

    Clausen, Michael Jakob

    unique expression profiles and specialized functional features. We use a Two Electrode Voltage Clamp setup to determine pre-steady-state and steady-state characteristics of each isoform and design chimeras to pin-point the structural elements responsible for observed differences. With this strategy we...... and glial cells express multiple isoforms of the Na+,K+-ATPase that are sorted to different specialized subcellular compartments. We are setting up a novel assay to study the details of Na+,K+-ATPase trafficking in polarized cells. With SNAP and CLIP tagged Na+,K+-ATPase isoforms we can track newly...

  16. FSH isoform pattern in classic galactosemia

    OpenAIRE

    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...

  17. PKC Isoform Expression in Modeled Microgravity

    Science.gov (United States)

    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.

  18. Isoform Specificity of Protein Kinase Cs in Synaptic Plasticity

    Science.gov (United States)

    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…

  19. Antiangiogenic VEGF Isoform in Inflammatory Myopathies

    Directory of Open Access Journals (Sweden)

    Nila Volpi

    2013-01-01

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

  20. Ikaros isoforms:The saga continues

    Institute of Scientific and Technical Information of China (English)

    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.

  1. New isoforms of rat Aquaporin-4

    DEFF Research Database (Denmark)

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

    2008-01-01

    an intracellular localization when expressed in cell lines and do not transport water when expressed in Xenopus oocytes. In contrast, the largest of the new isoforms, AQP4e, which contains a novel N-terminal domain, is localized at the plasma membrane in cell lines and functions as a water transporter in Xenopus...

  2. p53 isoforms change p53 paradigm

    OpenAIRE

    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.

  3. Isoforms of murine and human serum amyloid P component

    DEFF Research Database (Denmark)

    Nybo, Mads; Hackler, R; Kold, B;

    1998-01-01

    Isoelectric focusing (IEF) and immunofixation of murine serum amyloid P component (SAP), purified and in serum, showed a distinct and strain-dependent isoform pattern with up to seven bands (pI 5.1-5.7). Neuraminidase treatment caused a shift of the isoforms to more basic pI values, but did...... of isoforms of human SAP required the presence of urea and higher SAP concentrations. TEF and immunofixation of SAP monomers showed five to eight isoforms, ranging from pI 4.7-5.7. IEF of SAP in human serum resulted in a less distinct pattern and more acidic isoforms. As with murine SAP, neuraminidase...

  4. EASI—enrichment of alternatively spliced isoforms

    OpenAIRE

    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 ...

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

    DEFF Research Database (Denmark)

    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...

  6. Tumorigenic properties of alternative osteopontin isoforms in mesothelioma

    Energy Technology Data Exchange (ETDEWEB)

    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.

  7. Developmental expression of two Haliotis asinina hemocyanin isoforms.

    Science.gov (United States)

    Streit, Klaus; Jackson, Daniel; Degnan, Bernard M; Lieb, Bernhard

    2005-09-01

    Hemocyanins are large copper-containing respiratory proteins that play a role in oxygen transport in many molluscs. In some species only one hemocyanin isoform is present while in others two are expressed. The physiological relevance of these isoforms is unclear and the developmental and tissue-specific expression of hemocyanin genes is largely unknown. Here we show that two hemocyanin genes in the gastropod Haliotis asinina, which encode H. asinina hemocyanin (HaH1) and HaH2 isoforms, are developmentally expressed. These genes initially are expressed in a small number of mesenchyme cells at trochophore and pre-torsional veliger stages, with HaH1 expression slightly preceding HaH2. These cells largely are localized to the visceral mass, although a small number of cells are present in head and foot regions. Following metamorphosis the isoforms show overlapping as well as isoform-specific expression profiles, suggesting some degree of isoform-specific function.

  8. Androgen receptor isoforms in human and rat prostate

    Institute of Scientific and Technical Information of China (English)

    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)

  9. Evidence for leptin receptor isoforms heteromerization at the cell surface.

    Science.gov (United States)

    Bacart, Johan; Leloire, Audrey; Levoye, Angélique; Froguel, Philippe; Jockers, Ralf; Couturier, Cyril

    2010-06-01

    Leptin mediates its metabolic effects through several leptin receptor (LEP-R) isoforms. In humans, long (LEPRb) and short (LEPRa,c,d) isoforms are generated by alternative splicing. Most of leptin's effects are believed to be mediated by the OB-Rb isoform. However, the role of short LEPR isoforms and the possible existence of heteromers between different isoforms are poorly understood. Using BRET1 and optimized co-immunoprecipitation, we observed LEPRa/b and LEPRb/c heteromers located at the plasma membrane and stabilized by leptin. Given the widespread coexpression of LEPRa and LEPRb, our results suggest that LEPRa/b heteromers may represent a major receptor species in most tissues.

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

    Directory of Open Access Journals (Sweden)

    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.

  11. Cell-specific expression of TLR9 isoforms in inflammation.

    Science.gov (United States)

    McKelvey, Kelly J; Highton, John; Hessian, Paul A

    2011-02-01

    Toll-like receptors (TLRs) are key pattern recognition receptors during an immune response. With five isoforms of human TLR9 described, we hypothesised that differential expression of TLR9 isoforms in different cell types would result in variable contributions to the overall input from TLR9 during inflammation. We assessed the molecular expression of the TLR9 isoforms, TLR9-A, -C and -D. In normal peripheral blood mononuclear cells, B-lymphocytes express ∼100-fold more TLR9-A transcript than monocytes or T-lymphocytes, which predominantly express the TLR9-C transcript. Switches in isoform predominance accompany B-lymphocyte development. TLR9 protein expression in rheumatoid inflammatory lesions reflected the TLR9 isoform expression by immune cells. Herein we suggest that B-lymphocytes and plasmacytoid dendritic cells contribute the ∼3-fold higher TLR9-A transcript levels observed in inflamed synovium when compared to subcutaneous rheumatoid nodules. In contrast, macrophages and T-lymphocytes contribute the ∼4-fold higher TLR9-C transcript levels seen in nodules, compared to synovia. From protein sequence, predictions of subcellular localisation suggest TLR9-B may locate to the mitochondria, whereas TLR9-D adopts an opposing orientation in the endoplasmic reticulum. Consistent with this, structure models raise the possibility of alternative ligands for the TLR9-B and TLR9-D variants. Our results highlight differences in the expression of human TLR9 isoforms in normal and inflamed tissues, with differing contributions to inflammation.

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

    Directory of Open Access Journals (Sweden)

    Kubo Takeo

    2010-02-01

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

  13. Distinct functional interactions between actin isoforms and nonsarcomeric myosins.

    Directory of Open Access Journals (Sweden)

    Mirco Müller

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

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

    Science.gov (United States)

    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

  15. SURVIV for survival analysis of mRNA isoform variation.

    Science.gov (United States)

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

    2016-01-01

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

  16. Proteogenomic Analysis Identifies a Novel Human SHANK3 Isoform

    Directory of Open Access Journals (Sweden)

    Fahad Benthani

    2015-05-01

    Full Text Available Mutations of the SHANK3 gene have been associated with autism spectrum disorder. Individuals harboring different SHANK3 mutations display considerable heterogeneity in their cognitive impairment, likely due to the high SHANK3 transcriptional diversity. In this study, we report a novel interaction between the Mutated in colorectal cancer (MCC protein and a newly identified SHANK3 protein isoform in human colon cancer cells and mouse brain tissue. Hence, our proteogenomic analysis identifies a new human long isoform of the key synaptic protein SHANK3 that was not predicted by the human reference genome. Taken together, our findings describe a potential new role for MCC in neurons, a new human SHANK3 long isoform and, importantly, highlight the use of proteomic data towards the re-annotation of GC-rich genomic regions.

  17. Laminin isoforms in endothelial and perivascular basement membranes

    Science.gov (United States)

    Yousif, Lema F.; Di Russo, Jacopo; Sorokin, Lydia

    2013-01-01

    Laminins, one of the major functional components of basement membranes, are found underlying endothelium, and encasing pericytes and smooth muscle cells in the vessel wall. Depending on the type of blood vessel (capillary, venule, postcapillary venule, vein or artery) and their maturation state, both the endothelial and mural cell phenotype vary, with associated changes in laminin isoform expression. Laminins containing the α4 and α5 chains are the major isoforms found in the vessel wall, with the added contribution of laminin α2 in larger vessels. We here summarize current data on the precise localization of these laminin isoforms and their receptors in the different layers of the vessel wall, and their potential contribution to vascular homeostasis. PMID:23263631

  18. Oxygenation properties and isoform diversity of snake hemoglobins

    DEFF Research Database (Denmark)

    Storz, Jay F.; Natarajan, Chandrasekhar; Moriyama, Hideaki;

    2015-01-01

    Available data suggest that snake hemoglobins (Hbs) are characterized by a combination of unusual structural and functional properties relative to the Hbs of other amniote vertebrates, including oxygenation-linked tetramer- dimer dissociation. However, standardized comparative data are lacking...... for snake Hbs, and the Hb isoform composition of snake red blood cells has not been systematically characterized. Here we present the results of an integrated analysis of snake Hbs and the underlying - and -type globin genes to characterize 1) Hb isoform composition of definitive erythrocytes, and 2......) the oxygenation properties of isolated isoforms as well as composite hemolysates. We used species from three families as subjects for experimental studies of Hb function: South American rattlesnake, Crotalus durissus (Viperidae); Indian python, Python molurus (Pythonidae); and yellow-bellied sea snake, Pelamis...

  19. Identification and characterization of novel NuMA isoforms

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Jin, E-mail: petersdu2112@hotmail.com [Key Laboratory for Cell Proliferation and Regulation of the Ministry of Education, Beijing Normal University, Beijing (China); Xu, Zhe [Department of Clinical Laboratory Diagnosis, Beijing Tiantan Hospital, Capital Medical University, Beijing (China); Core Laboratory for Clinical Medical Research, Beijing Tiantan Hospital, Capital Medical University, Beijing (China); He, Dacheng [Key Laboratory for Cell Proliferation and Regulation of the Ministry of Education, Beijing Normal University, Beijing (China); Lu, Guanting, E-mail: guantlv@126.com [Beijing DnaLead Science and Technology Co., LTD, Beijing (China)

    2014-11-21

    Highlights: • Seven NuMA isoforms generated by alternative splicing were categorized into 3 groups: long, middle and short. • Both exons 15 and 16 in long NuMA were “hotspot” for alternative splicing. • Lower expression of short NuMA was observed in cancer cells compared with nonneoplastic controls. • Distinct localization pattern of short isoforms indicated different function from that of long and middle NuMA. - Abstract: The large nuclear mitotic apparatus (NuMA) has been investigated for over 30 years with functions related to the formation and maintenance of mitotic spindle poles during mitosis. However, the existence and functions of NuMA isoforms generated by alternative splicing remains unclear. In the present work, we show that at least seven NuMA isoforms (categorized into long, middle and short groups) generated by alternative splicing from a common NuMA mRNA precursor were discovered in HeLa cells and these isoforms differ mainly at the carboxyl terminus and the coiled-coil domains. Two “hotspot” exons with molecular mass of 3366-nt and 42-nt tend to be spliced during alternative splicing in long and middle groups. Furthermore, full-length coding sequences of long and middle NuMA obtained by using fusion PCR were constructed into GFP-tagged vector to illustrate their cellular localization. Long NuMA mainly localized in the nucleus with absence from nucleoli during interphase and translocated to the spindle poles in mitosis. Middle NuMA displayed the similar cell cycle-dependent distribution pattern as long NuMA. However, expression of NuMA short isoforms revealed a distinct subcellular localization. Short NuMA were present in the cytosol during the whole cycle, without colocalization with mitotic apparatus. These results have allowed us tentatively to explore a new research direction for NuMA’s various functions.

  20. A Review of Metallothionein Isoforms and their Role in Pathophysiology

    OpenAIRE

    Senthil kumar M; Manisenthil Kumar KT; Shyam Sunder A; Thirumoorthy N; Ganesh GNK; Chatterjee Malay

    2011-01-01

    Abstract The Metallothionein (MT) is a protein which has several interesting biological effects and has been demonstrated increase focus on the role of MT in various biological systems in the past three decades. The studies on the role of MT were limited with few areas like apoptosis and antioxidants in selected organs even fifty years after its discovery. Now acknowledge the exploration of various isoforms of MT such as MT-I, MT-II, MT-III and MT-IV and other isoforms in various biological s...

  1. ApoE isoform-dependent changes in hippocampal synaptic function

    Directory of Open Access Journals (Sweden)

    Sullivan Patrick M

    2009-05-01

    Full Text Available Abstract The lipoprotein receptor system in the hippocampus is intimately involved in the modulation of synaptic transmission and plasticity. The association of specific apoE isoform expression with human neurodegenerative disorders has focused attention on the role of these apoE isoforms in lipoprotein receptor-dependent synaptic modulation. In the present study, we used the apoE2, apoE3 and apoE4 targeted replacement (TR mice along with recombinant human apoE isoforms to determine the role of apoE isoforms in hippocampus area CA1 synaptic function. While synaptic transmission is unaffected by apoE isoform, long-term potentiation (LTP is significantly enhanced in apoE4 TR mice versus apoE2 TR mice. ApoE isoform-dependent differences in LTP induction require NMDA-receptor function, and apoE isoform expression alters activation of both ERK and JNK signal transduction. Acute application of specific apoE isoforms also alters LTP induction while decreasing NMDA-receptor mediated field potentials. Furthermore, acute apoE isoform application does not have the same effects on ERK and JNK activation. These findings demonstrate specific, isoform-dependent effects of human apoE isoforms on adult hippocampus synaptic plasticity and highlight mechanistic differences between chronic apoE isoform expression and acute apoE isoform exposure.

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

    Science.gov (United States)

    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.

  3. Bacteria-Induced Dscam Isoforms of the Crustacean, Pacifastacus leniusculus.

    Directory of Open Access Journals (Sweden)

    Apiruck Watthanasurorot

    2011-06-01

    Full Text Available The Down syndrome cell adhesion molecule, also known as Dscam, is a member of the immunoglobulin super family. Dscam plays an essential function in neuronal wiring and appears to be involved in innate immune reactions in insects. The deduced amino acid sequence of Dscam in the crustacean Pacifastacus leniusculus (PlDscam, encodes 9(Ig-4(FNIII-(Ig-2(FNIII-TM and it has variable regions in the N-terminal half of Ig2 and Ig3 and the complete Ig7 and in the transmembrane domain. The cytoplasmic tail can generate multiple isoforms. PlDscam can generate more than 22,000 different unique isoforms. Bacteria and LPS injection enhanced the expression of PlDscam, but no response in expression occurred after a white spot syndrome virus (WSSV infection or injection with peptidoglycans. Furthermore, PlDscam silencing did not have any effect on the replication of the WSSV. Bacterial specific isoforms of PlDscam were shown to have a specific binding property to each tested bacteria, E. coli or S. aureus. The bacteria specific isoforms of PlDscam were shown to be associated with bacterial clearance and phagocytosis in crayfish.

  4. Role of p53 isoforms and aggregations in cancer.

    Science.gov (United States)

    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

  5. Identification and characterization of a novel isoform of hepatopoietin

    Institute of Scientific and Technical Information of China (English)

    Jun Lu; Wang-Xiang Xu; Yi-Qun Zhan; Xiao-Lin Cui; Wei-Min Cai; Fu-Chu He; Xiao-Ming Yang

    2002-01-01

    AIM: To isolate a novel isoform of human HPO (HPO-205)human fetal liver Marathon-reedy cDNA andcharacterize its primary biological function.METHODS: 5'-RACE (rapid amplification of cDNA 5' ends)was used to isolate a novel isoform of hHPO in this paperThe constructed pcDNAHPO-205, pcDNAHPO and pcDNA eukaryotic expression vectors were respectively transfectedby lipofectamine method and the stimulation of DNAsynthesis was observed by 3H-TdR incorporation assay.Proteins extracted from different cells were analyzed byWestern blot.RESULTS: A novel isoform of hHPO (HPO-205) encoding a205 amino acid ORF corresponding to a translatedproduction of 23 kDa was isolated and distinguished fromthe previous HPO that lacked the N-terminal 80 amino acids.The dnse-dspendent stimulation of DNA synthesis of HepG2hepatoma cells by HPO-205 demonstrated its similarbiological activity with HPO in vitro. The level of MAPK(Mitogen-activated protein kinase) phnsphorylarion byWestern blot analysis revealed that HPO-205 might have thestronger activity of stimulating hepatic cell proliferation thanthat of HPO.CONCLUSION: A novel isoform of hHPO (HPO-205) wasisolated from hepatic-derived cells. The comparison of HPO-205 and HPO will lead to a new insight into the structure andfunction of hHPO, and provide the new way of thinking todeeply elucidate the biological roles of HPO/ALR.

  6. Distinct Functions of Endophilin Isoforms in Synaptic Vesicle Endocytosis

    Directory of Open Access Journals (Sweden)

    Jifeng Zhang

    2015-01-01

    Full Text Available Endophilin isoforms perform distinct characteristics in their interactions with N-type Ca2+ channels and dynamin. However, precise functional differences for the endophilin isoforms on synaptic vesicle (SV endocytosis remain unknown. By coupling RNA interference and electrophysiological recording techniques in cultured rat hippocampal neurons, we investigated the functional differences of three isoforms of endophilin in SV endocytosis. The results showed that the amplitude of normalized evoked excitatory postsynaptic currents in endophilin1 knockdown neurons decreased significantly for both single train and multiple train stimulations. Similar results were found using endophilin2 knockdown neurons, whereas endophilin3 siRNA exhibited no change compared with control neurons. Endophilin1 and endophilin2 affected SV endocytosis, but the effect of endophilin1 and endophilin2 double knockdown was not different from that of either knockdown alone. This result suggested that endophilin1 and endophilin2 functioned together but not independently during SV endocytosis. Taken together, our results indicate that SV endocytosis is sustained by endophilin1 and endophilin2 isoforms, but not by endophilin3, in primary cultured hippocampal neurons.

  7. Isoforms of transferrin in psoriasis patients abusing alcohol

    NARCIS (Netherlands)

    P. Hoefkens (Peter); E.M. Higgins; R.J. Ward (Roberta); H.G. van Eijk (Henk)

    1997-01-01

    textabstractThe different isoforms of transferrin have been quantified by isoelectric focusing in the sera of psoriasis patients with and without a history of abusing alcohol. In both male and female psoriasis subjects abusing alcohol, there were significant increases in the 2-sial

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

    Science.gov (United States)

    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

  9. Cloning, expression and alternative splicing of the novel isoform of hTCP11 gene

    DEFF Research Database (Denmark)

    Ma, Yong-xin; Zhang, Si-zhong; Wu, Qia-qing;

    2003-01-01

    To identify a novel isoform of hTCP11 gene and investigate its expression and alternative splicing.......To identify a novel isoform of hTCP11 gene and investigate its expression and alternative splicing....

  10. Growth hormone isoforms in a girl with gigantism.

    Science.gov (United States)

    Ng, L L; Chasalow, F I; Escobar, O; Blethen, S L

    1999-01-01

    Several previous investigations have suggested that there may be different growth hormone isoforms in patients with acromegaly. We used three different site-specific monoclonal antibodies (MAbs) to investigate growth hormone (GH) isoforms in serum from an 8 year-old girl with a GH and prolactin secreting adenoma. The pattern of GH-immunoreactivity was dependent on the circumstances of collection. Serum obtained after oral glucose had very little cross reactivity with MAb 352 although concentrations of up to 15 micrograms/l were found with two other MAbs, 033 and 665. MAb 352 does not recognize the 20,000 dalton isoform of GH (20K) while both MAb 033 and 665 do. The same pattern of GH immunoreactivity (low MAb 352, equal and higher MAb 033 and 665) was seen in other baseline samples. In contrast, samples obtained after TRH/GnRH showed immunoreactivity patterns expected for a mixture of 22,000 dalton isoform of GH (22K) with only a small amount of 20K. GH samples obtained during sleep showed both patterns with episodic peaks with equal immunoreactivity superimposed on the basal pattern (decreased activity with MAb 352). Affinity chromatography of basal samples showed that a portion of the GH immunoreactivity was neither 22K nor 20K, although in stimulated samples, over 70% of GH was 22K or 20K GH. In conclusion, the nature of GH isoforms present in serum varies with GH concentration. These differences may contribute to the known difficulty in correlating disease activity and random GH measurements in patients with GH secreting adenomas. PMID:10392356

  11. Determination of the class and isoform selectivity of small-molecule histone deacetylase inhibitors

    DEFF Research Database (Denmark)

    Khan, N.; Jeffers, M.; Kumar, S.;

    2008-01-01

    ) against a panel of rhHDAC (recombinant human HDAC) isoforms. Eight rhHDACs were expressed using a baculoviral system, and a Fluor de Lystrade mark (Biomol International) HDAC assay was optimized for each purified isoform. The potency and selectivity of ten HDACs on class I isoforms (rhHDAC1, rhHDAC2, rh...

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

    DEFF Research Database (Denmark)

    Windahl, Michael Skovbo; Boesen, Jane; Karlsen, Pernille Efferbach;

    2009-01-01

    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...

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  14. EGFR soluble isoforms and their transcripts are expressed in meningiomas.

    Science.gov (United States)

    Guillaudeau, Angélique; Durand, Karine; Bessette, Barbara; Chaunavel, Alain; Pommepuy, Isabelle; Projetti, Fabrice; Robert, Sandrine; Caire, François; Rabinovitch-Chable, Hélène; Labrousse, François

    2012-01-01

    The EGFR (epidermal growth factor receptor) is involved in the oncogenesis of many tumors. In addition to the full-length EGFR (isoform a), normal and tumor cells produce soluble EGFR isoforms (sEGFR) that lack the intracellular domain. sEGFR isoforms b, c and d are encoded by EGFR variants 2 (v2), 3 (v3) and 4 (v4) mRNA resulting from gene alternative splicing. Accordingly, the results of EGFR protein expression analysis depend on the domain targeted by the antibodies. In meningiomas, EGFR expression investigations mainly focused on EGFR isoform a. sEGFR and EGFRvIII mutant, that encodes a constitutively active truncated receptor, have not been studied. In a 69 meningiomas series, protein expression was analyzed by immunohistochemistry using extracellular domain targeted antibody (ECD-Ab) and intracellular domain targeted antibody (ICD-Ab). EGFRv1 to v4 and EGFRvIII mRNAs were quantified by RT-PCR and EGFR amplification revealed by MLPA. Results were analyzed with respect to clinical data, tumor resection (Simpson grade), histological type, tumor grade, and patient outcome.Immunochemical staining was stronger with ECD-Ab than with ICD-Ab. Meningiomas expressed EGFRv1 to -v4 mRNAs but not EGFRvIII mutant. Intermediate or high ECD-Ab staining and high EGFRv1 to v4 mRNA levels were associated to a better progression free survival (PFS). PFS was also improved in women, when tumor resection was evaluated as Simpson 1 or 2, in grade I vs. grade II and III meningiomas and when Ki67 labeling index was lower than 10%. Our results suggest that, EGFR protein isoforms without ICD and their corresponding mRNA variants are expressed in meningiomas in addition to the whole isoform a. EGFRvIII was not expressed. High expression levels seem to be related to a better prognosis. These results indicate that the oncogenetic mechanisms involving the EGFR pathway in meningiomas could be different from other tumor types. PMID:22623992

  15. EGFR soluble isoforms and their transcripts are expressed in meningiomas.

    Directory of Open Access Journals (Sweden)

    Angélique Guillaudeau

    Full Text Available The EGFR (epidermal growth factor receptor is involved in the oncogenesis of many tumors. In addition to the full-length EGFR (isoform a, normal and tumor cells produce soluble EGFR isoforms (sEGFR that lack the intracellular domain. sEGFR isoforms b, c and d are encoded by EGFR variants 2 (v2, 3 (v3 and 4 (v4 mRNA resulting from gene alternative splicing. Accordingly, the results of EGFR protein expression analysis depend on the domain targeted by the antibodies. In meningiomas, EGFR expression investigations mainly focused on EGFR isoform a. sEGFR and EGFRvIII mutant, that encodes a constitutively active truncated receptor, have not been studied. In a 69 meningiomas series, protein expression was analyzed by immunohistochemistry using extracellular domain targeted antibody (ECD-Ab and intracellular domain targeted antibody (ICD-Ab. EGFRv1 to v4 and EGFRvIII mRNAs were quantified by RT-PCR and EGFR amplification revealed by MLPA. Results were analyzed with respect to clinical data, tumor resection (Simpson grade, histological type, tumor grade, and patient outcome.Immunochemical staining was stronger with ECD-Ab than with ICD-Ab. Meningiomas expressed EGFRv1 to -v4 mRNAs but not EGFRvIII mutant. Intermediate or high ECD-Ab staining and high EGFRv1 to v4 mRNA levels were associated to a better progression free survival (PFS. PFS was also improved in women, when tumor resection was evaluated as Simpson 1 or 2, in grade I vs. grade II and III meningiomas and when Ki67 labeling index was lower than 10%. Our results suggest that, EGFR protein isoforms without ICD and their corresponding mRNA variants are expressed in meningiomas in addition to the whole isoform a. EGFRvIII was not expressed. High expression levels seem to be related to a better prognosis. These results indicate that the oncogenetic mechanisms involving the EGFR pathway in meningiomas could be different from other tumor types.

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

    Directory of Open Access Journals (Sweden)

    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.

  17. Characterization of four hemocyanin isoforms in Litopenaeus vannamei

    Institute of Scientific and Technical Information of China (English)

    XU Jingxiang; RUAN Lingwei; LI Zhen; YU Xiaoman; LI Sedong; SHI Hong; XU Xun

    2015-01-01

    In this study, the gene encoding hemocyanin subunit L, LvHcL, was cloned from Litopenaeus vannamei and the genomic organization was characterized. This gene was diverse with many SNPs and also had at least four isoforms, while one of them (LvHcL4) only had two exons and the exon2 was missed. Transcription analysis showed that these isoforms of LvHcL were up-regulated after WSSV challenge in WSSV-resistant shrimp, while the transcriptions were decreased constantly in WSSV-susceptible shrimp. It is suggested that the hemocyanin had rich polymorphism and was involved in the antiviral response. These results could extend our previous findings and provide insights into the immune feature of hemocyanin, which would be helpful for further studies aimed at antiviral mechanism in inver-tebrate.

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

    OpenAIRE

    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 ...

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

    OpenAIRE

    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...

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

    Directory of Open Access Journals (Sweden)

    Ralph D Hector

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

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

    Science.gov (United States)

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

    2016-01-01

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

  2. Antagonistic functions of two stardust isoforms in Drosophila photoreceptor cells.

    Science.gov (United States)

    Bulgakova, Natalia A; Rentsch, Michaela; Knust, Elisabeth

    2010-11-15

    Membrane-associated guanylate kinases (MAGUKs) are scaffolding proteins that organize supramolecular protein complexes, thereby partitioning the plasma membrane into spatially and functionally distinct subdomains. Their modular organization is ideally suited to organize protein complexes with cell type- or stage-specific composition, or both. Often more than one MAGUK isoform is expressed by one gene in the same cell, yet very little is known about their individual in vivo functions. Here, we show that two isoforms of Drosophila stardust, Sdt-H (formerly called Sdt-B2) and Sdt-D, which differ in their N terminus, are expressed in adult photoreceptors. Both isoforms associate with Crumbs and PATJ, constituents of the conserved Crumbs-Stardust complex. However, they form distinct complexes, localized at the stalk, a restricted region of the apical plasma membrane. Strikingly, Sdt-H and Sdt-D have antagonistic functions. While Sdt-H overexpression increases stalk membrane length and prevents light-dependent retinal degeneration, Sdt-D overexpression reduces stalk length and enhances light-dependent retinal degeneration. These results suggest that a fine-tuned balance of different Crumbs complexes regulates photoreceptor homeostasis.

  3. Characterisation of CDKL5 Transcript Isoforms in Human and Mouse

    Science.gov (United States)

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

    2016-01-01

    Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5) cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3’-untranslated region (UTR), which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders. PMID:27315173

  4. Molecular mechanical differences between isoforms of contractile actin in the presence of isoforms of smooth muscle tropomyosin.

    OpenAIRE

    Lennart Hilbert; Genevieve Bates; Roman, Horia N.; Jenna L Blumenthal; Zitouni, Nedjma B.; Apolinary Sobieszek; Mackey, Michael C.; Anne-Marie Lauzon

    2013-01-01

    The proteins involved in smooth muscle's molecular contractile mechanism - the anti-parallel motion of actin and myosin filaments driven by myosin heads interacting with actin - are found as different isoforms. While their expression levels are altered in disease states, their relevance to the mechanical interaction of myosin with actin is not sufficiently understood. Here, we analyzed in vitro actin filament propulsion by smooth muscle myosin for [Formula: see text]-actin ([Formula: see text...

  5. Caspase-8 regulates TNF-alpha-induced epithelial necroptosis and terminal ileitis

    OpenAIRE

    Günther, Claudia; Martini, Eva; Wittkopf, Nadine; Amann, Kerstin; Weigmann, Benno; Neumann, Helmut; Waldner, Maximilian J.; Hedrick, Stephen M; Tenzer, Stefan; Neurath, Markus F; Becker, Christoph

    2011-01-01

    Dysfunction of the intestinal epithelium is believed to result in excessive translocation of commensal bacteria into the bowel wall that drives chronic mucosal inflammation in Crohn's disease; an incurable inflammatory bowel disease in humans characterized by inflammation of the terminal ileum 1 . Beside the physical barrier established by the tight contact of cells, specialized epithelial cells such as Paneth cells and goblet cells provide innate immune defence functions by secreting mucus a...

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

    Science.gov (United States)

    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

  7. PKC isoforms interact with and phosphorylate DNMT1

    Directory of Open Access Journals (Sweden)

    Pradhan Sriharsa

    2011-05-01

    Full Text Available Abstract Background DNA methyltransferase 1 (DNMT1 has been shown to be phosphorylated on multiple serine and threonine residues, based on cell type and physiological conditions. Although recent studies have suggested that protein kinase C (PKC may be involved, the individual contribution of PKC isoforms in their ability to phosphorylate DNMT1 remains unknown. The PKC family consists of at least 12 isoforms that possess distinct differences in structure, substrate requirement, expression and localization. Results Here we show that PKCα, βI, βII, δ, γ, η, ζ and μ preferentially phosphorylate the N-terminal domain of human DNMT1. No such phosphorylation of DNMT1 was observed with PKCε. Using PKCζ as a prototype model, we also found that PKC physically interacts with and phosphorylates DNMT1. In vitro phosphorylation assays conducted with recombinant fragments of DNMT1 showed that PKCζ preferentially phosphorylated the N-terminal region of DNMT1. The interaction of PKCζ with DNMT1 was confirmed by GST pull-down and co-immunoprecipitation experiments. Co-localization experiments by fluorescent microscopy further showed that endogenous PKCζ and DNMT1 were present in the same molecular complex. Endogenous PKCζ activity was also detected when DNMT1 was immunoprecipitated from HEK-293 cells. Overexpression of both PKCζ and DNMT1 in HEK-293 cells, but not of either alone, reduced the methylation status of genes distributed across the genome. Moreover, in vitro phosphorylation of DNMT1 by PKCζ reduced its methytransferase activity. Conclusions Our results indicate that phosphorylation of human DNMT1 by PKC is isoform-specific and provides the first evidence of cooperation between PKCζ and DNMT1 in the control of the DNA methylation patterns of the genome.

  8. The FU gene and its possible protein isoforms

    Directory of Open Access Journals (Sweden)

    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

  9. Functional differences between L- and T-plastin isoforms

    OpenAIRE

    1994-01-01

    Fimbrins/plastins are a family of highly conserved actin-bundling proteins. They are present in all eukaryotic cells including yeast, but each isoform displays a remarkable tissue specificity. T-plastin is normally found in epithelial and mesenchymal cells while L-plastin is present in hematopoietic cells. However, L-plastin has been also found in tumor cells of non-hematopoietic origin (Lin, C.-S., R. H. Aebersold, S. B. Kent, M. Varma, and J. Leavitt. 1988. Mol. Cell. Biol. 8:4659-4668; Lin...

  10. Soluble malate dehydrogenase of Geophagus brasiliensis (Cichlidae, Perciformes: isolated isoforms and kinetics properties

    Directory of Open Access Journals (Sweden)

    Maria Regina de Aquino-Silva

    2008-01-01

    Full Text Available Kinetic properties and thermal stabilities of Geophagus brasiliensis skeletal muscle unfractionated malate dehydrogenase (MDH, EC 1.1.1.37 and its isolated isoforms were analyzed to examine a possible sMDH-B* locus duplication in a fixation process influenced by genetic drift. Two optimal pHs were detected: 7.5 for AB1 unfractionated muscle phenotype and its B1 isoform, and 8.0 for AB1B2 unfractionated muscle phenotype, A and B2 isoforms. While G. brasiliensis A isoform could be characterized as thermostable, the duplicated B isoform cannot be assumed as thermolabile. Km values for isolated B2 isoforms were 1.6 times lower than for B1. A duplication event in progress best explains the electrophoretic six-band pattern detected in G. brasiliensis, which would be caused by genetic drift.

  11. Induction of Chemokine Expression by Adiponectin In Vitro is Isoform-Dependent

    OpenAIRE

    Song, Huijuan; Chan, James; Rovin, Brad H.

    2009-01-01

    Adiponectin is reported to have both pro- and anti-inflammatory effects. Because adiponectin circulates in isoforms of various sizes, and some responses to adiponectin are isoform-dependent, it was postulated that the pro-inflammatory effects of adiponectin may isoform-specific. To test this, peripheral blood mononuclear cells (PBMC), microvascular endothelial cells (MVEC), and human glomerular mesangial cells (HMC) were treated with high or low molecular weight (HMW, LMW) recombinant human a...

  12. MetaDiff: differential isoform expression analysis using random-effects meta-regression

    OpenAIRE

    Jia, Cheng; Guan, Weihua; Yang, Amy; Xiao, Rui; Tang, W. H. Wilson; Moravec, Christine S.; Margulies, Kenneth B.; Cappola, Thomas P.; Li, Mingyao; Li, Chun

    2015-01-01

    Background RNA sequencing (RNA-Seq) allows an unbiased survey of the entire transcriptome in a high-throughput manner. A major application of RNA-Seq is to detect differential isoform expression across experimental conditions, which is of great biological interest due to its direct relevance to protein function and disease pathogenesis. Detection of differential isoform expression is challenging because of uncertainty in isoform expression estimation owing to ambiguous reads and variability i...

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

    OpenAIRE

    Wu, I; Shin, S. C.; Cao, Y; Bender, I K; N Jafari; 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 expres...

  14. IsoformEx: isoform level gene expression estimation using weighted non-negative least squares from mRNA-Seq data

    Directory of Open Access Journals (Sweden)

    Gupta Ravi

    2011-07-01

    Full Text Available Abstract Background mRNA-Seq technology has revolutionized the field of transcriptomics for identification and quantification of gene transcripts not only at gene level but also at isoform level. Estimating the expression levels of transcript isoforms from mRNA-Seq data is a challenging problem due to the presence of constitutive exons. Results We propose a novel algorithm (IsoformEx that employs weighted non-negative least squares estimation method to estimate the expression levels of transcript isoforms. Validations based on in silico simulation of mRNA-Seq and qRT-PCR experiments with real mRNA-Seq data showed that IsoformEx could accurately estimate transcript expression levels. In comparisons with published methods, the transcript expression levels estimated by IsoformEx showed higher correlation with known transcript expression levels from simulated mRNA-Seq data, and higher agreement with qRT-PCR measurements of specific transcripts for real mRNA-Seq data. Conclusions IsoformEx is a fast and accurate algorithm to estimate transcript expression levels and gene expression levels, which takes into account short exons and alternative exons with a weighting scheme. The software is available at http://bioinformatics.wistar.upenn.edu/isoformex.

  15. Prostaglandin D Synthase Isoforms from Cerebrospinal Fluid Vary with Brain Pathology

    Directory of Open Access Journals (Sweden)

    Michael G. Harrington

    2006-01-01

    Full Text Available Glutathione independent prostaglandin D synthase (Swissprot P41222, PTGDS has been identified in human cerebrospinal fluid and some changes in PTGDS in relation to disease have been reported. However, little is known of the extent that PTGDS isoforms fluctuate across a large range of congenital and acquired diseases. The purpose of this study was to examine changes in PTGDS isoforms in such a population. Spinal fluid from 22 healthy study participants (normal controls with no classifiable neurological or psychiatric diagnosis was obtained and PTGDS isoforms were identified by specific immunostaining and mass spectrometry after denaturing 2D gel electrophoresis. The PTGDS isoforms in controls consisted of five charge isoforms that were always present and a small number of occasional, low abundance isoforms. A qualitative survey of 98 different people with a wide range of congenital and acquired diseases revealed striking changes. Loss of the control isoforms occurred in congenital malformations of the nervous system. Gain of additional isoforms occurred in some degenerative, most demyelinating and vasculitic diseases, as well as in Creutzfeldt-Jakob disease. A retrospective analysis of published data that quantified relative amounts of PTGDS in multiple sclerosis, schizophrenia and Parkinson’s disease compared to controls revealed significant dysregulation. It is concluded that qualitative and quantitative fluctuations of cerebrospinal fluid PTGDS isoforms reflect both major and subtle brain pathophysiology.

  16. Proteomic Analysis of Cytokeratin Isoforms Uncovers Association with Survival in Lung Adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Tarek G. Gharib

    2002-01-01

    Full Text Available Cytokeratins. (CK are intermediate filaments whose expression is often altered in epithelial cancer. Systematic identification of lung adenocarcinoma proteins using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry has uncovered numerous CK isoforms. In this study, 93 lung adenocarcinomas. (64 stage I and 29 stage III and 10 uninvolved lung samples were quantitatively examined for protein expression. Fourteen of 21 isoforms of CK 7, 8, 18, 19 occurred at significantly higher levels. (P<.05 in tumors compared to uninvolved adjacent tissue. Specific isoforms of the four types of CK identified correlated with either clinical outcome or individual clinical-pathological parameters. All five of the CK7 isoforms associated with patient survival represented cleavage products. Two of five CK7 isoforms. (nos. 2165 and 2091, one of eight CK8 isoforms. (no. 439, one of three CK19 isoforms. (no. 1955 were associated with survival and significantly correlated to their mRNA levels, suggesting that transcription underlies overexpression of these CK isoforms. Our data indicate substantial heterogeneity among CK in lung adenocarcinomas resulting from posttranslational modifications, some of which correlated with patient survival and other clinical parameters. Therefore, specific isoforms of individual CK may have utility as diagnostic or predictive markers in lung adenocarcinomas.

  17. Entropy-based model for miRNA isoform analysis.

    Directory of Open Access Journals (Sweden)

    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.

  18. A New View of Ras Isoforms in Cancers.

    Science.gov (United States)

    Nussinov, Ruth; Tsai, Chung-Jung; Chakrabarti, Mayukh; Jang, Hyunbum

    2016-01-01

    Does small GTPase K-Ras4A have a single state or two states, one resembling K-Ras4B and the other N-Ras? A recent study of K-Ras4A made the remarkable observation that even in the absence of the palmitoyl, K-Ras4A can be active at the plasma membrane. Importantly, this suggests that K-Ras4A may exist in two distinct signaling states. In state 1, K-Ras4A is only farnesylated, like K-Ras4B; in state 2, farnesylated and palmitoylated, like N-Ras. The K-Ras4A hypervariable region sequence is positively charged, in between K-Ras4B and N-Ras. Taken together, this raises the possibility that the farnesylated but nonpalmitoylated state 1, like K-Ras4B, binds calmodulin and is associated with colorectal and other adenocarcinomas like lung cancer and pancreatic ductal adenocarcinoma. On the other hand, state 2 may be associated with melanoma and other cancers where N-Ras is a major contributor, such as acute myeloid leukemia. Importantly, H-Ras has two, singly and doubly, palmitoylated states that may also serve distinct functional roles. The multiple signaling states of palmitoylated Ras isoforms question the completeness of small GTPase Ras isoform statistics in different cancer types and call for reevaluation of concepts and protocols. They may also call for reconsideration of oncogenic Ras therapeutics. PMID:26659836

  19. A Review of Metallothionein Isoforms and their Role in Pathophysiology

    Directory of Open Access Journals (Sweden)

    Senthil kumar M

    2011-05-01

    Full Text Available Abstract The Metallothionein (MT is a protein which has several interesting biological effects and has been demonstrated increase focus on the role of MT in various biological systems in the past three decades. The studies on the role of MT were limited with few areas like apoptosis and antioxidants in selected organs even fifty years after its discovery. Now acknowledge the exploration of various isoforms of MT such as MT-I, MT-II, MT-III and MT-IV and other isoforms in various biological systems. Strong evidence exists that MT modulates complex diseases and the immune system in the body but the primary function of MT still remains unknown. This review's main objective is to explore the capability to specifically manipulate MT levels in cells and in animals to provide answers regarding how MT could impact those complex disease scenarios. The experimental result mentioned in this review related among MT, zinc, cadmium, diabetic, heart disease, bone retardation, neuro toxicity, kidney dysfunction, cancer, and brain suggest novel method for exploration and contribute significantly to the growing scientist to research further in this field.

  20. Structures of alternatively spliced isoforms of human ketohexokinase.

    Science.gov (United States)

    Trinh, Chi H; Asipu, Aruna; Bonthron, David T; Phillips, Simon E V

    2009-03-01

    A molecular understanding of the unique aspects of dietary fructose metabolism may be the key to understanding and controlling the current epidemic of fructose-related obesity, diabetes and related adverse metabolic states in Western populations. Fructose catabolism is initiated by its phosphorylation to fructose 1-phosphate, which is performed by ketohexokinase (KHK). Here, the crystal structures of the two alternatively spliced isoforms of human ketohexokinase, hepatic KHK-C and the peripheral isoform KHK-A, and of the ternary complex of KHK-A with the substrate fructose and AMP-PNP are reported. The structure of the KHK-A ternary complex revealed an active site with both the substrate fructose and the ATP analogue in positions ready for phosphorylation following a reaction mechanism similar to that of the pfkB family of carbohydrate kinases. Hepatic KHK deficiency causes the benign disorder essential fructosuria. The effects of the disease-causing mutations (Gly40Arg and Ala43Thr) have been modelled in the context of the KHK structure.

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

    Science.gov (United States)

    Silver, Kristopher S; Soderlund, David M

    2006-07-15

    Pyrazoline-type insecticides are potent inhibitors of insect and mammalian voltage-sensitive sodium channels. In mammals, there are nine sodium channel alpha subunit isoforms that have unique distributions and pharmacological properties, but no published data exist that compare the relative sensitivity of these different mammalian sodium channel isoforms to inhibition by pyrazoline-type insecticides. This study employed the Xenopus oocyte expression system to examine the relative sensitivity of rat Na(v)1.2a, Na(v)1.4, Na(v)1.5, and Na(v)1.8 sodium channel alpha subunit isoforms to the pyrazoline-type insecticides indoxacarb, DCJW, and RH 3421. Additionally, we assessed the effect of coexpression with the rat beta1 auxiliary subunit on the sensitivity of the Na(v)1.2a and Na(v)1.4 isoforms to these compounds. The relative sensitivity of the four sodium channel alpha subunits differed for each of the three compounds we examined. With DCJW, the order of sensitivity was Na(v)1.4 > Na(v)1.2a > Na(v)1.5 > Na(v)1.8. In contrast, the relative sensitivity of these isoforms to indoxacarb differed from that to DCJW: the Na(v)1.8 isoform was most sensitive, the Na(v)1.4 isoform was completely insensitive, and the sensitivities of the Na(v)1.5 and Na(v)1.2a isoforms were intermediate between these two extremes. Moreover, the pattern of sensitivity to RH 3421 among these four isoforms was different from that for either indoxacarb or DCJW: the Na(v)1.4 isoform was most sensitive to RH 3421, whereas the sensitivities of the remaining three isoforms were substantially less than that of the Na(v)1.4 isoform and were approximately equivalent. The only statistically significant effect of coexpression of either the Na(v)1.2a or Na(v)1.4 isoforms with the beta1 subunit was the modest reduction in the sensitivity of the Na(v)1.2a isoform to RH 3421. These results demonstrate that mammalian sodium channel isoforms differ in their sensitivities to pyrazoline-type insecticides.

  2. Expression of phosphoinositide-specific phospholipase C isoforms in native endothelial cells.

    Directory of Open Access Journals (Sweden)

    Delphine M Béziau

    Full Text Available Phospholipase C (PLC comprises a superfamily of enzymes that play a key role in a wide array of intracellular signalling pathways, including protein kinase C and intracellular calcium. Thirteen different mammalian PLC isoforms have been identified and classified into 6 families (PLC-β, γ, δ, ε, ζ and η based on their biochemical properties. Although the expression of PLC isoforms is tissue-specific, concomitant expression of different PLC has been reported, suggesting that PLC family is involved in multiple cellular functions. Despite their critical role, the PLC isoforms expressed in native endothelial cells (ECs remains undetermined. A conventional PCR approach was initially used to elucidate the mRNA expression pattern of PLC isoforms in 3 distinct murine vascular beds: mesenteric (MA, pulmonary (PA and middle cerebral arteries (MCA. mRNA encoding for most PLC isoforms was detected in MA, MCA and PA with the exception of η2 and β2 (only expressed in PA, δ4 (only expressed in MCA, η1 (expressed in all but MA and ζ (not detected in any vascular beds tested. The endothelial-specific PLC expression was then sought in freshly isolated ECs. Interestingly, the PLC expression profile appears to differ across the investigated arterial beds. While mRNA for 8 of the 13 PLC isoforms was detected in ECs from MA, two additional PLC isoforms were detected in ECs from PA and MCA. Co-expression of multiple PLC isoforms in ECs suggests an elaborate network of signalling pathways: PLC isoforms may contribute to the complexity or diversity of signalling by their selective localization in cellular microdomains. However in situ immunofluorescence revealed a homogeneous distribution for all PLC isoforms probed (β3, γ2 and δ1 in intact endothelium. Although PLC isoforms play a crucial role in endothelial signal transduction, subcellular localization alone does not appear to be sufficient to determine the role of PLC in the signalling microdomains found

  3. Nesprins: tissue-specific expression of epsilon and other short isoforms.

    Directory of Open Access Journals (Sweden)

    Nguyen Thuy Duong

    Full Text Available Nesprin-1-giant and nesprin-2-giant regulate nuclear positioning by the interaction of their C-terminal KASH domains with nuclear membrane SUN proteins and their N-terminal calponin-homology domains with cytoskeletal actin. A number of short isoforms lacking the actin-binding domains are produced by internal promotion. We have evaluated the significance of these shorter isoforms using quantitative RT-PCR and western blotting with site-specific monoclonal antibodies. Within a complete map of nesprin isoforms, we describe two novel nesprin-2 epsilon isoforms for the first time. Epsilon isoforms are similar in size and structure to nesprin-1-alpha. Expression of nesprin isoforms was highly tissue-dependent. Nesprin-2-epsilon-1 was found in early embryonic cells, while nesprin-2-epsilon-2 was present in heart and other adult tissues, but not skeletal muscle. Some cell lines lack shorter isoforms and express only one of the two nesprin genes, suggesting that either of the giant nesprins is sufficient for basic cell functions. For the first time, localisation of endogenous nesprin away from the nuclear membrane was shown in cells where removal of the KASH domain by alternative splicing occurs. By distinguishing between degradation products and true isoforms on western blots, it was found that previously-described beta and gamma isoforms are expressed either at only low levels or with a limited tissue distribution. Two of the shortest alpha isoforms, nesprin-1-alpha-2 and nesprin-2-alpha-1, were found almost exclusively in cardiac and skeletal muscle and a highly conserved and alternatively-spliced exon, available in both nesprin genes, was always included in these tissues. These "muscle-specific" isoforms are thought to form a complex with emerin and lamin A/C at the inner nuclear membrane and mutations in all three proteins cause Emery-Dreifuss muscular dystrophy and/or inherited dilated cardiomyopathy, disorders in which only skeletal muscle and

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

    Science.gov (United States)

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

    2016-01-01

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

  5. Glial fibrillary acidic protein isoform expression in plaque related astrogliosis in Alzheimer's disease.

    Science.gov (United States)

    Kamphuis, Willem; Middeldorp, Jinte; Kooijman, Lieneke; Sluijs, Jacqueline A; Kooi, Evert-Jan; Moeton, Martina; Freriks, Michel; Mizee, Mark R; Hol, Elly M

    2014-03-01

    In Alzheimer's disease (AD), amyloid plaques are surrounded by reactive astrocytes with an increased expression of intermediate filaments including glial fibrillary acidic protein (GFAP). Different GFAP isoforms have been identified that are differentially expressed by specific subpopulations of astrocytes and that impose different properties to the intermediate filament network. We studied transcript levels and protein expression patterns of all known GFAP isoforms in human hippocampal AD tissue at different stages of the disease. Ten different transcripts for GFAP isoforms were detected at different abundancies. Transcript levels of most isoforms increased with AD progression. GFAPδ-immunopositive astrocytes were observed in subgranular zone, hilus, and stratum-lacunosum-moleculare. GFAPδ-positive cells also stained for GFAPα. In AD donors, astrocytes near plaques displayed increased staining of both GFAPα and GFAPδ. The reading-frame-shifted isoform, GFAP(+1), staining was confined to a subset of astrocytes with long processes, and their number increased in the course of AD. In conclusion, the various GFAP isoforms show differential transcript levels and are upregulated in a concerted manner in AD. The GFAP(+1) isoform defines a unique subset of astrocytes, with numbers increasing with AD progression. These data indicate the need for future exploration of underlying mechanisms concerning the functions of GFAPδ and GFAP(+1) isoforms in astrocytes and their possible role in AD pathology.

  6. Activation of AMPK alpha and gamma-isoform complexes in the intact ischemic rat heart

    Science.gov (United States)

    AMP-activated protein kinase (AMPK) plays a key role in modulating cellular metabolic processes. AMPK, a serine-threonine kinase, is a heterotrimeric complex of catalytic alpha-subunits and regulatory beta- and gamma-subunits with multiple isoforms. Mutations in the cardiac gamma(2)-isoform have bee...

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

    Science.gov (United States)

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

    2016-04-01

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

  8. Molecular cloning and pharmacology of functionally distinct isoforms of the human histamine H(3) receptor

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Goodman, M W; Burstein, E S;

    2002-01-01

    The pharmacology of histamine H(3) receptors suggests the presence of distinct receptor isoforms or subtypes. We herein describe multiple, functionally distinct, alternatively spliced isoforms of the human H(3) receptor. Combinatorial splicing at three different sites creates at least six distinc...

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

    Science.gov (United States)

    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

  10. Roles of the troponin isoforms during indirect flight muscle development in Drosophila

    Indian Academy of Sciences (India)

    Salam Herojeet Singh; Prabodh Kumar; Nallur B. Ramachandra; Upendra Nongthomba

    2014-08-01

    Troponin proteins in cooperative interaction with tropomyosin are responsible for controlling the contraction of the striated muscles in response to changes in the intracellular calcium concentration. Contractility of the muscle is determined by the constituent protein isoforms, and the isoforms can switch over from one form to another depending on physiological demands and pathological conditions. In Drosophila, amajority of themyofibrillar proteins in the indirect flight muscles (IFMs) undergo post-transcriptional and post-translational isoform changes during pupal to adult metamorphosis to meet the high energy and mechanical demands of flight. Using a newly generated Gal4 strain (UH3-Gal4) which is expressed exclusively in the IFMs, during later stages of development, we have looked at the developmental and functional importance of each of the troponin subunits (troponin-I, troponin-T and troponin-C) and their isoforms. We show that all the troponin subunits are required for normal myofibril assembly and flight, except for the troponin-C isoform 1 (TnC1). Moreover, rescue experiments conducted with troponin-I embryonic isoform in the IFMs, where flies were rendered flightless, show developmental and functional differences of TnI isoforms and importance of maintaining the right isoform.

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

    Science.gov (United States)

    AN ENZYME LINKED IMMUNOSORBENT ASSAY FOR THE HO-1 ISOFORM OF HEME OXYGENASE Heme oxygenase (HO) occurs in biological tissues as two major isoforms HO-1 and HO-2. HO-1 is inducible by many treatments, particularly oxidative stress-related conditions such as depletion of gl...

  12. Translational control of C/EBPalpha and C/EBPbeta isoform expression

    NARCIS (Netherlands)

    Calkhoven, C F; Müller, C; Leutz, A

    2000-01-01

    Transcription factors derived from CCAAT/enhancer binding protein (C/EBP)alpha and C/EBPbeta genes control differentiation and proliferation in a number of cell types. Various C/EBP isoforms arise from unique C/EBPbeta and C/EBPalpha mRNAs by differential initiation of translation. These isoforms re

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

    Science.gov (United States)

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

    2016-04-01

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

  14. Recombinant erythropoietin in humans has a prolonged effect on circulating erythropoietin isoform distribution

    DEFF Research Database (Denmark)

    Aachmann-Andersen, Niels Jacob; Just Christensen, Søren; Lisbjerg, Kristian;

    2014-01-01

    -acetyl glucosamine with the glycosylation dependent desorption of EPO isoforms. At day 25, plasma-EPO in both rhEPO groups had returned to values not different from the placebo group. PMI with placebo, reflecting the endogenous EPO isoforms, averaged 82.5 (10.3) % (mean (SD)). High-dose Epoetin beta decreased PMI...

  15. Differences in expression, actions and cocaine regulation of two isoforms for the brain transcriptional regulator NAC1.

    Science.gov (United States)

    Korutla, L; Wang, P J; Lewis, D M; Neustadter, J H; Stromberg, M F; Mackler, S A

    2002-01-01

    BTB/POZ proteins can influence the cell cycle and contribute to oncogenesis. Many family members are present in the mammalian CNS. Previous work demonstrated elevated NAC1 mRNA levels in the rat nucleus accumbens in response to cocaine. NAC1 acts like other BTB/POZ proteins that regulate transcription but is unusual because of the absence of identifiable DNA binding domains. cDNAs were isolated encoding two NAC1 isoforms differing by only 27 amino acids (the longer isoform contains 514 amino acids). The mRNAs for both isoforms were simultaneously expressed throughout the rat brain and peripheral tissues. Semi-quantitative reverse transcription-polymerase chain reaction analysis revealed that the mRNA of the longer isoform was more abundant than the mRNA of the shorter isoform. Western blot analysis demonstrated a similar unequal distribution between the isoforms in the CNS. The longer isoform was the more abundant of the two NAC1 proteins and the ratio between them differed throughout the rat brain. The shorter isoform was not detected in most of the examined peripheral tissues, suggesting differences from the CNS in post-transcriptional processing. Both isoforms repressed transcription in H293T cells using a Gal4-luciferase reporter system. However, the shorter isoform did not repress transcription as effectively as the longer isoform. Transfection of different ratios for both isoforms, in order to replicate the relative amounts observed throughout the CNS, supported an interaction between the isoforms. The net effect on transcriptional repression was determined by the ratio of the two NAC1 isoforms. Each isoform exhibited the subnuclear localization that is characteristic of many BTB/POZ proteins. A rapid and transient increase in the level of the shorter isoform occurred in the nucleus accumbens 2 h following a single i.p. cocaine injection. We conclude that the two isoforms of NAC1 may differentially affect neuronal functions, including the regulation of

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

    Science.gov (United States)

    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

  17. Troponin T isoform expression is modulated during Atlantic Halibut metamorphosis

    Directory of Open Access Journals (Sweden)

    Llewellyn Lynda

    2007-06-01

    Full Text Available Abstract Background Flatfish metamorphosis is a thyroid hormone (TH driven process which leads to a dramatic change from a symmetrical larva to an asymmetrical juvenile. The effect of THs on muscle and in particular muscle sarcomer protein genes is largely unexplored in fish. The change in Troponin T (TnT, a pivotal protein in the assembly of skeletal muscles sarcomeres and a modulator of calcium driven muscle contraction, during flatfish metamophosis is studied. Results In the present study five cDNAs for halibut TnT genes were cloned; three were splice variants arising from a single fast TnT (fTnT gene; a fourth encoded a novel teleost specific fTnT-like cDNA (AfTnT expressed exclusively in slow muscle and the fifth encoded the teleost specific sTnT2. THs modified the expression of halibut fTnT isoforms which changed from predominantly basic to acidic isoforms during natural and T4 induced metamorphosis. In contrast, expression of red muscle specific genes, AfTnT and sTnT2, did not change during natural metamorphosis or after T4 treatment. Prior to and after metamorphosis no change in the dorso-ventral symmetry or temporal-spatial expression pattern of TnT genes and muscle fibre organization occurred in halibut musculature. Conclusion Muscle organisation in halibut remains symmetrical even after metamorphosis suggesting TH driven changes are associated with molecular adaptations. We hypothesize that species specific differences in TnT gene expression in teleosts underlies different larval muscle developmental programs which better adapts them to the specific ecological constraints.

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

    Science.gov (United States)

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

    1999-01-01

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

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

    Science.gov (United States)

    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

  20. Effect of proteasome inhibitors on expression of HLA-G isoforms.

    Science.gov (United States)

    Poláková, K; Bandzuchová, E; Bystrická, M; Pancuchárová, H; Russ, G

    2006-01-01

    HLA-G primary transcript is alternatively spliced into a number of mRNAs. In addition to full length HLA-G1 protein isoform these mRNAs might also encode truncated HLA-G protein isoforms lacking one or two extracellular domains. Whereas HLA-G1 protein isoform is regularly identified, truncated HLAG protein isoforms are not detected even if all alternative spliced mRNAs are present in cells. The absence of entire domain(s) renders the truncated HLA-G protein isoforms incapable of binding peptide and beta2-microglobulin. These features of truncated HLA-G protein isoforms may result in their rapid degradation by proteasomes. Here we show that despite the presence of all alternatively spliced HLA-G transcripts in JEG-3 cells pretreated with proteasome inhibitors only a full length HLA-G1 protein isoform was regularly detected. Interestingly, immunoblot analysis showed slight increase of HLA-G1 protein in cells pretreated with proteasome inhibitors, although the expression of HLA-G1 transcript was basically not affected. Expression of HLA-G3 transcript increased in JEG-3 cells pre-incubated with LLL, however, neither HLA-G3 nor other HLA-G short protein isoform was regularly detected. In K562 transfectants proteasome inhibitor LLL greatly enhanced expression of the HLA-G1 and -G2 transcripts as well as corresponding protein isoforms. Flow cytometry analysis showed that in cells pre-treated with proteasome inhibitors cell surface expression of HLA-G1 protein decreased but the quantity of intracellularly localized HLA-G antigens increased. Altogether our results suggest that truncated HLA-G proteins isoforms are not detected in JEG-3 cells as a result of their instability and the low translation efficiency of truncated HLA-G transcripts.

  1. Characterization of ductal and lobular breast carcinomas using novel prolactin receptor isoform specific antibodies

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    Heger Christopher D

    2010-12-01

    Full Text Available Abstract Background Prolactin is a polypeptide hormone responsible for proliferation and differentiation of the mammary gland. More recently, prolactin's role in mammary carcinogenesis has been studied with greater interest. Studies from our laboratory and from others have demonstrated that three specific isoforms of the prolactin receptor (PRLR are expressed in both normal and cancerous breast cells and tissues. Until now, reliable isoform specific antibodies have been lacking. We have prepared and characterized polyclonal antibodies against each of the human PRLR isoforms that can effectively be used to characterize human breast cancers. Methods Rabbits were immunized with synthetic peptides of isoform unique regions and immune sera affinity purified prior to validation by Western blot and immunohistochemical analyses. Sections of ductal and lobular carcinomas were stained with each affinity purified isoform specific antibody to determine expression patterns in breast cancer subclasses. Results We show that the rabbit antibodies have high titer and could specifically recognize each isoform of PRLR. Differences in PRLR isoform expression levels were observed and quantified using histosections from xenografts of established human breast cancer cells lines, and ductal and lobular carcinoma human biopsy specimens. In addition, these results were verified by real-time PCR with isoform specific primers. While nearly all tumors contained LF and SF1b, the majority (76% of ductal carcinoma biopsies expressed SF1a while the majority of lobular carcinomas lacked SF1a staining (72% and 27% had only low levels of expression. Conclusions Differences in the receptor isoform expression profiles may be critical to understanding the role of PRL in mammary tumorigenesis. Since these antibodies are specifically directed against each PRLR isoform, they are valuable tools for the evaluation of breast cancer PRLR content and have potential clinical importance in

  2. Androgen receptor isoforms in human prostatic cancer tissue and LNCaP cell line

    Institute of Scientific and Technical Information of China (English)

    Shu-Jie XIA; Xiao-Da TANG; Qing-Zheng MA

    2001-01-01

    Aim: To investigate the androgen receptor (AR) isoform expressions in human prostatic cancer tissue and LNCaP cell line. Methods: With high resolution isoelectric focusing (IEF) method we demonstrated the different expressions of AR isoforms in human prostatic cancer tissues and LNCaP cell line. Results: Data were obtained from three prostatic cancer specimens and the LNCaP cell line. Three types of AR isoforms were detected with pI values at 6.5,6.0, and 5.3. For the 3 prostatic cancer specimens, 1 sample showed all the three types of AR isoforms, the second specimen expressed at 6.5 and 6.0, and the third failed to show any type of isoforms. The LNCaP cell line expressed all the three AR isoforms. Binding of 3H-dihydrotestosterone (3H-DHT) to these three isoforms was inhibited by the addition ofl00-fold excess of DHT or testosterone, while not by progesterone, oestradiol and diethylstilboestrol. Conclusion: The expression of AR isofonns is different in different prostate cancer tissues, which may be related to the difference in the effect of anti-androgen therapy in different patients.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Estrogen and progesterone receptor isoforms expression in the stomach of Mongolian gerbils

    Institute of Scientific and Technical Information of China (English)

    Milena Saqui-Salces; Teresa Neri-Gómez; Armando Gamboa-Dominguez; Guillermo Ruiz-Palacios; Ignacio Camacho-Arroyo

    2008-01-01

    AIM:We studied the estrogen receptor (ER) and progesterone receptor (PR) isoforms expression in gastric antrum and corpus of female gerbils and their regulation by estradiol (E2) and progesterone (P4).METHODS: Ovariectomized adult female gerbils were subcutaneously treated with E2,and E2 + P4.Uteri and stomachs were removed,the latter were cut along the greater curvature,and antrum and corpus were excised.Proteins were immunoblotted using antibodies that recognize ER-alpha,ER-beta,and PR-A and PR-B receptor isoforms.Tissues from rats treated in the same way were used as controls.RESULTS: Specific bands were detected for ER-alpha (68 KDa),and PR isoforms (85 and 120 KDa for PR-A and PR-B isoforrns,respectively) in uteri,gastric antrum and corpus.We could not detect ER-beta isoform.PR isoforms were not regulated by E2 or P4 in uterus and gastric tissues of gerbils.ER-alpha isoform content was significantly down-regulated by E2 in the corpus,but not affected by hormones in uterus and gastric antrum.CONCLUSION: The presence of ER-alpha and PR isoforms in gerbils stomach suggests that E2 and P4 actions in this organ are in part mediated by their nuclear receptors.

  6. Quarternary structure and enzymological properties of the different hormone-sensitive lipase (HSL isoforms.

    Directory of Open Access Journals (Sweden)

    Christian Krintel

    Full Text Available BACKGROUND: Hormone-sensitive lipase (HSL is a key enzyme in the mobilization of energy in the form of fatty acids from intracellular stores of neutral lipids. The enzyme has been shown to exist in different isoforms with different molecular masses (84 kDa, 89 kDa and 117 kDa expressed in a tissue-dependent manner, where the predominant 84 kDa form in adipocytes is the most extensively studied. METHODOLOGY/PRINCIPAL FINDINGS: In this study we employed negative stain electron microscopy (EM to analyze the quarternary structure of the different HSL isoforms. The results show that all three isoforms adopt a head-to-head homodimeric organization, where each monomer contains two structural domains. We also used enzymatic assays to show that despite the variation in the size of the N-terminal domain all three isoforms exhibit similar enzymological properties with regard to psychrotolerance and protein kinase A (PKA-mediated phosphorylation and activation. CONCLUSIONS/SIGNIFICANCE: We present the first data on the quaternary structure and domain organization of the three HSL isoforms. We conclude that despite large differences in the size of the N-terminal, non-catalytic domain all three HSL isoforms exhibit the same three-dimensional architecture. Furthermore, the three HSL isoforms are very similar with regard to two unique enzymological characteristics of HSL, i.e., cold adaptation and PKA-mediated activation.

  7. Dynamic expression and localization of c-MET isoforms in the developing rat pancreas.

    Science.gov (United States)

    Wu, Yulong; Cheng, Mei; Shi, Zhen; Feng, Zhenqing; Guan, Xiaohong

    2014-01-01

    Pancreata from Sprague Dawley rats of different developmental stages were studied to determine the expression and cellular localization of different c-MET isoforms in the developing rat pancreas. Pancreatic mRNA and protein expression levels of c-MET at different developmental stages from embryo to adult were detected by reverse transcription-polymerase chain reaction and by western blotting. To identify the cellular localization of c-MET protein in the developing rat pancreas, double immunofluorescent staining was performed using antibodies for cell type-specific markers and for c-MET. The expression of two isoforms of c-MET (190 kDa and 170 kDa) coincided with the development of the pancreas. The 190 kDa isoform of c-MET is expressed during embryonic stages, and its expression is replaced by the expression of the 170 kDa isoform as the pancreas develops. Only the 170 kDa isoform is expressed in the adult rat pancreas. Throughout all stages of pancreatic development, c-MET is expressed by vimentin-positive cells. In contrast, c-MET staining was stronger in rat pancreata from newborn to adult stages and overlapped with insulin-positive beta-cells. The dynamic expression and localization of different c-MET isoforms in the rat pancreas during different developmental stages indicates that distinct c-MET isoform might be involved in different aspects of pancreatic development.

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

    Science.gov (United States)

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

    2014-03-15

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

  9. The isolation of parvalbumin isoforms from the tail muscle of the American alligator (Alligator mississipiensis).

    Science.gov (United States)

    Laney, E L; Shabanowitz, J; King, G; Hunt, D F; Nelson, D J

    1997-04-01

    Multiple parvalbumin isoforms have been detected in the tail (skeletal) muscle of the American alligator (Alligator mississipiensis). One of these isoforms (APV-1) has been highly purified and partially characterized. Protein purification involved mainly gel filtration and anion exchange chromatography, and characterization included gel electrophoresis, amino acid composition analysis, metal ion analysis, MALDI-TOF and ESI mass spectrometry, ultraviolet and fluorescence spectroscopy, and one- and two-dimensional 500 MHz proton NMR spectroscopy. The alligator isoforms are rich in phenylalanine and deficient in the other aromatic residues as is typical for parvalbumins. In fact, the one highly purified isoform that forms the basis of this study has only phenyl-alanine as an aromatic residue. Ion exchange chromatography further indicates that this isoform has a relatively high isoelectric point (pl approximately 5.0), indicating that it is an alpha-lineage parvalbumin. This alligator parvalbumin isoform is unusual in that it has an atypically high Ca2+ content (almost 3.0 mole of Ca2+ per mole of protein) following purification, a fact supported by terbium fluorescence titration experiments. Preliminary comparative analysis of the highly purified alligator parvalbumin isoform (in the Ca2-loaded state) by two-dimensional 1H-NMR (2D 1H TOCSY and 2D 1H NOESY) indicates that there is considerable similarity in structure between the alligator protein and a homologous protein obtained from the silver hake (a saltwater fish species). PMID:9076974

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

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

    2013-10-01

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

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

    Science.gov (United States)

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

    2013-01-01

    Muscle fiber type is a well studied property in limb muscles, however, much less is understood about myosin heavy chain (MHC) isoform expression in caudal muscles of mammalian tails. Didelphid marsupials are an interesting lineage in this context as all species have prehensile tails, but show a range of tail-function depending on either their arboreal or terrestrial locomotor habits. Differences in prehensility suggest that MHC isoform fiber types may also be different, in that terrestrial opossums may have a large distribution of oxidative fibers for object carrying tasks instead of faster, glycolytic fiber types expected in mammals with long tails. To test this hypothesis, MHC isoform fiber type and their regional distribution (proximal/transitional/distal) were determined in the tail of the Virginia opossum (Didelphis virginiana). Fiber types were determined by a combination of myosin-ATPase histochemistry, immunohistochemistry, and SDS-PAGE. Results indicate a predominance of the fast MHC-2A and -2X isoforms in each region of the tail. The presence of two fast isoforms, in addition to the slow MHC-1 isoform, was confirmed by SDS-PAGE analysis. The overall MHC isoform fiber type distribution for the tail was: 25% MHC-1, 71% MHC-2A/X hybrid, and 4% MHC-1/2A hybrid. Oxidative MHC-2A/X isoform fibers were found to be relatively large in cross-section compared to slow, oxidative MHC-1 and MHC-1/2A hybrid fibers. A large percentage of fast MHC-2A/X hybrids fibers may be suggestive of an evolutionary transition in MHC isoform distribution (fast-to-slow fiber type) in the tail musculature of an opossum with primarily a terrestrial locomotor habit and adaptive tail-function. PMID:23152195

  13. Isoform-specific upregulation of palladin in human and murine pancreas tumors.

    Directory of Open Access Journals (Sweden)

    Silvia M Goicoechea

    Full Text Available Pancreatic ductal adenocarcinoma (PDA is a lethal disease with a characteristic pattern of early metastasis, which is driving a search for biomarkers that can be used to detect the cancer at an early stage. Recently, the actin-associated protein palladin was identified as a candidate biomarker when it was shown that palladin is mutated in a rare inherited form of PDA, and overexpressed in many sporadic pancreas tumors and premalignant precursors. In this study, we analyzed the expression of palladin isoforms in murine and human PDA and explored palladin's potential use in diagnosing PDA. We performed immunohistochemistry and immunoblot analyses on patient samples and tumor-derived cells using an isoform-selective monoclonal antibody and a pan-palladin polyclonal antibody. Immunoblot and real-time quantitative reverse transcription-PCR were used to quantify palladin mRNA levels in human samples. We show that there are two major palladin isoforms expressed in pancreas: 65 and 85-90 kDa. The 65 kDa isoform is expressed in both normal and neoplastic ductal epithelial cells. The 85-90 kDa palladin isoform is highly overexpressed in tumor-associated fibroblasts (TAFs in both primary and metastatic tumors compared to normal pancreas, in samples obtained from either human patients or genetically engineered mice. In tumor-derived cultured cells, expression of palladin isoforms follows cell-type specific patterns, with the 85-90 kDa isoform in TAFs, and the 65 kDa isoform predominating in normal and neoplastic epithelial cells. These results suggest that upregulation of 85-90 kDa palladin isoform may play a role in the establishment of the TAF phenotype, and thus in the formation of a desmoplastic tumor microenvironment. Thus, palladin may have a potential use in the early diagnosis of PDA and may have much broader significance in understanding metastatic behavior.

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

    Science.gov (United States)

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

    2013-01-01

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

  15. Purification and Characterization of Two Voltage-Dependent Anion Channel Isoforms from Plant Seeds1

    Science.gov (United States)

    Abrecht, Helge; Wattiez, Ruddy; Ruysschaert, Jean-Marie; Homblé, Fabrice

    2000-01-01

    Mitochondria were isolated from imbibed seeds of lentil (Lens culinaris) and Phaseolus vulgaris. We copurified two voltage-dependent anion channel from detergent solubilized mitochondria in a single purification step using hydroxyapatite. The two isoforms from P. vulgaris were separated by chromatofocusing chromatography in 4 m urea without any loss of channel activity. Channel activity of each isoform was characterized upon reconstitution into diphytanoyl phosphatidylcholine planar lipid bilayers. Both isoforms form large conductance channels that are slightly anion selective and display cation selective substates. PMID:11080295

  16. Purification and characterization of two voltage-dependent anion channel isoforms from plant seeds.

    Science.gov (United States)

    Abrecht, H; Wattiez, R; Ruysschaert, J M; Homblé, F

    2000-11-01

    Mitochondria were isolated from imbibed seeds of lentil (Lens culinaris) and Phaseolus vulgaris. We copurified two voltage-dependent anion channel from detergent solubilized mitochondria in a single purification step using hydroxyapatite. The two isoforms from P. vulgaris were separated by chromatofocusing chromatography in 4 M urea without any loss of channel activity. Channel activity of each isoform was characterized upon reconstitution into diphytanoyl phosphatidylcholine planar lipid bilayers. Both isoforms form large conductance channels that are slightly anion selective and display cation selective substates.

  17. Mitochondrial localization of the OAS1 p46 isoform associated with a common single nucleotide polymorphism

    DEFF Research Database (Denmark)

    Kjær, Karina Hansen; Pahus, Jytte; Hansen, Mariann Fagernæs;

    2014-01-01

    genotypes in the OAS1 SNP rs10774671, HeLa cells with the AA genotype, HT1080 cells with AG, and Daudi cells with GG. The main OAS1 isoform expressed in Daudi and HT1080 cells was p46, and the main OAS1 isoform expressed in HeLa cells was p42. In addition, low levels of the OAS1 p52 mRNA was detected in He...... vacuoles/lysosomes. By using recombinantly expressed OAS1 mutant proteins, we found that the OAS1 SNP rs1131454 (former rs3741981) did not affect the enzymatic OAS1 activity. Conclusions: The SNP rs10774671 determines differential expression of the OAS1 isoforms. In Daudi and HT1080 cells the p46 isoform...

  18. Glial fibrillary acidic protein isoform expression in plaque related astrogliosis in Alzheimer's disease

    NARCIS (Netherlands)

    Kamphuis, W.; Middeldorp, Jinte; Kooijman, Lieneke; Sluijs, Jacqueline A; Kooi, Evert-Jan; Moeton, Martina; Freriks, Michel; Mizee, Mark R; Hol, Elly M

    2014-01-01

    In Alzheimer's disease (AD), amyloid plaques are surrounded by reactive astrocytes with an increased expression of intermediate filaments including glial fibrillary acidic protein (GFAP). Different GFAP isoforms have been identified that are differentially expressed by specific subpopulations of ast

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

    Science.gov (United States)

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

    1999-01-01

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

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

    DEFF Research Database (Denmark)

    Song, Lina; Bachert, Collin; Schjoldager, Katrine T;

    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....... Surprisingly, molecular probes to monitor GalNAc-transferase activity are lacking and there exist no effective global or isoform-specific inhibitors. Here we describe the development of T2- and T3-isoform specific fluorescence sensors that traffic in the secretory pathway. Each sensor yielded little signal...... in both the study of GalNAc-transferase regulation and in high-throughput screening for potential therapeutic regulators of specific GalNAc-transferases....

  1. Receptor-isoform-selective insulin analogues give tissue-preferential effects

    DEFF Research Database (Denmark)

    Vienberg, Sara Gry; Bouman, Stephan D; Sørensen, Heidi;

    2011-01-01

    The relative expression patterns of the two IR (insulin receptor) isoforms, +/- exon 11 (IR-B/IR-A respectively), are tissue-dependent. Therefore we have developed insulin analogues with different binding affinities for the two isoforms to test whether tissue-preferential biological effects can...... be attained. In rats and mice, IR-B is the most prominent isoform in the liver (> 95%) and fat (> 90%), whereas in muscles IR-A is the dominant isoform (> 95%). As a consequence, the insulin analogue INS-A, which has a higher relative affinity for human IR-A, had a higher relative potency [compared with HI...... (human insulin)] for glycogen synthesis in rat muscle strips (26%) than for glycogen accumulation in rat hepatocytes (5%) and for lipogenesis in rat adipocytes (4%). In contrast, the INS-B analogue, which has an increased affinity for human IR-B, had higher relative potencies (compared with HI...

  2. VAMP/synaptobrevin isoforms 1 and 2 are widely and differentially expressed in nonneuronal tissues

    Science.gov (United States)

    1996-01-01

    VAMP/synaptobrevin is part of the synaptic vesicle docking and fusion complex and plays a central role in neuroexocytosis. Two VAMP (vesicle- associated membrane protein) isoforms are expressed in the nervous system and are differently distributed among the specialized parts of the tissue. Here, VAMP-1 and -2 are shown to be present in all rat tissues tested, including kidney, adrenal gland, liver, pancreas, thyroid, heart, and smooth muscle. The two isoforms are differentially expressed in various tissues and their level may depend on differentiation. VAMP-1 is restricted to exocrine pancreas and to kidney tubular cells, whereas VAMP-2 is the predominant isoform present in Langerhans islets and in glomerular cells. Both isoforms show a patchy vesicular intracellular distribution in confocal microscopy. The present results provide evidence for the importance of neuronal VAMP proteins in the physiology of all cells. PMID:8567721

  3. The cytoplasmic 60 kDa progesterone receptor isoform predominates in the human amniochorion and placenta at term

    Directory of Open Access Journals (Sweden)

    Bell Stephen C

    2009-03-01

    Full Text Available Abstract Background The mechanism that initiates human parturition has been proposed to be 'functional progesterone withdrawal' whereby the 116 kDa B-isoform of the progesterone receptor (PR-B switches in favour of the 94 kDa A-isoform (PR-A in reproductive tissues. Recently, other PR isoforms, PR-S, PR-C and PR-M generated from the same gene have been identified and partially characterised. Methods and Results Using immunohistochemical, western blotting and RT-PCR techniques, evidence is provided that indicates the major PR isoform present in human term fetal membranes (amnion and chorion and syncytiotrophoblast of the placenta is neither of the classical nuclear PR-B or PR-A isoforms but is the N-terminally truncated 60 kDa PR-C isoform. Evidence is also provided that this 60 kDa isoform resides in the cytoplasm of the expressing cell types. Data are also presented to show that PR-B, PR-A and PR-S isoforms are essentially absent from the amnion and chorion, whereas PR isoforms A, B, C and S are all present in the decidua, with PR-A being the major isoform. The syncytiotrophoblast of the placenta contains the cytoplasmic 60 kDa isoform, but not isoforms PR-A, PR-B or PR-S. Conclusion The major PR isoform in the amnion, chorion and placenta is a 60 kDa protein that could be PR-C, suggesting that the cytoplasmic isoform has a specific role in extra-embryonic tissues and may be involved in the regulation of human parturition.

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

    Science.gov (United States)

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

    2016-07-01

    Tropomyosin (Tpm) is an α helical coiled-coil dimer that forms a co-polymer along the actin filament. Tpm is involved in the regulation of actin's interaction with binding proteins as well as stabilization of the actin filament and its assembly kinetics. Recent studies show that multiple Tpm isoforms also define the functional properties of distinct actin filament populations within a cell. Subtle structural variations within well conserved Tpm isoforms are the key to their functional specificity. Therefore, we purified and characterized a comprehensive set of 8 Tpm isoforms (Tpm1.1, Tpm1.12, Tpm1.6, Tpm1.7, Tpm1.8, Tpm2.1, Tpm3.1, and Tpm4.2), using well-established actin co-sedimentation and pyrene fluorescence polymerization assays. We observed that the apparent affinity (Kd(app)) to filamentous actin varied in all Tpm isoforms between ∼0.1-5 μM with similar values for both, skeletal and cytoskeletal actin filaments. The data did not indicate any correlation between affinity and size of Tpm molecules, however high molecular weight (HMW) isoforms Tpm1.1, Tpm1.6, Tpm1.7 and Tpm2.1, showed ∼3-fold higher cooperativity compared to low molecular weight (LMW) isoforms Tpm1.12, Tpm1.8, Tpm3.1, and Tpm4.2. The rate of actin filament elongation in the presence of Tpm2.1 increased, while all other isoforms decreased the elongation rate by 27-85 %. Our study shows that the biochemical properties of Tpm isoforms are finely tuned and depend on sequence variations in alternatively spliced regions of Tpm molecules. PMID:27420374

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

    OpenAIRE

    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...

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

    OpenAIRE

    Velten, Brandy P.; Welch, Kenneth C.

    2014-01-01

    Myosin heavy chain (MHC) isoform complement is intimately related to a muscle's contractile properties, yet relatively little is known about avian MHC isoforms or how they may vary with fiber type and/or the contractile properties of a muscle. The rapid shortening of muscles necessary to power flight at the high wingbeat frequencies of ruby-throated hummingbirds and zebra finches (25–60 Hz), along with the varied morphology and use of the hummingbird hindlimb, provides a unique opportunity to...

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

    OpenAIRE

    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...

  8. Recombinant erythropoietin in humans has a prolonged effect on circulating erythropoietin isoform distribution.

    Directory of Open Access Journals (Sweden)

    Niels Jacob Aachmann-Andersen

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

  9. Genomic organization and the tissue distribution of alternatively spliced isoforms of the mouse Spatial gene

    Directory of Open Access Journals (Sweden)

    Mattei Marie-Geneviève

    2004-07-01

    Full Text Available Abstract Background The stromal component of the thymic microenvironment is critical for T lymphocyte generation. Thymocyte differentiation involves a cascade of coordinated stromal genes controlling thymocyte survival, lineage commitment and selection. The "Stromal Protein Associated with Thymii And Lymph-node" (Spatial gene encodes a putative transcription factor which may be involved in T-cell development. In the testis, the Spatial gene is also expressed by round spermatids during spermatogenesis. Results The Spatial gene maps to the B3-B4 region of murine chromosome 10 corresponding to the human syntenic region 10q22.1. The mouse Spatial genomic DNA is organised into 10 exons and is alternatively spliced to generate two short isoforms (Spatial-α and -γ and two other long isoforms (Spatial-δ and -ε comprising 5 additional exons on the 3' site. Here, we report the cloning of a new short isoform, Spatial-β, which differs from other isoforms by an additional alternative exon of 69 bases. This new exon encodes an interesting proline-rich signature that could confer to the 34 kDa Spatial-β protein a particular function. By quantitative TaqMan RT-PCR, we have shown that the short isoforms are highly expressed in the thymus while the long isoforms are highly expressed in the testis. We further examined the inter-species conservation of Spatial between several mammals and identified that the protein which is rich in proline and positive amino acids, is highly conserved. Conclusions The Spatial gene generates at least five alternative spliced variants: three short isoforms (Spatial-α, -β and -γ highly expressed in the thymus and two long isoforms (Spatial-δ and -ε highly expressed in the testis. These alternative spliced variants could have a tissue specific function.

  10. Kinetics of local and systemic isoforms of serum amyloid A in bovine mastitic milk

    DEFF Research Database (Denmark)

    Jacobsen, Stine; Niewold, T.A.; Kornalijnslijper, E.;

    2005-01-01

    The aim of the present study was to characterise the serum amyloid A (SAA) response to intramammary inoculation of Escherichia coli and to examine the distribution of hepatically and extrahepatically pruduced SAA isoforms in plasma and milk fra cows with mastitis.......The aim of the present study was to characterise the serum amyloid A (SAA) response to intramammary inoculation of Escherichia coli and to examine the distribution of hepatically and extrahepatically pruduced SAA isoforms in plasma and milk fra cows with mastitis....

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

    Science.gov (United States)

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

    2016-07-01

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

  12. Opposing effects of fructokinase C and A isoforms on fructose-induced metabolic syndrome in mice

    OpenAIRE

    Ishimoto, Takuji; Lanaspa, Miguel A.; MyPhuong T Le; Garcia, Gabriela E.; Diggle, Christine P; MacLean, Paul S.; Jackman, Matthew R.; Asipu, Aruna; Roncal-Jimenez, Carlos A.; Kosugi, Tomoki; Rivard, Christopher J.; Maruyama, Shoichi; Rodriguez-Iturbe, Bernardo; Sánchez-Lozada, Laura G.; Bonthron, David T.

    2012-01-01

    Fructose intake from added sugars correlates with the epidemic rise in obesity, metabolic syndrome, and nonalcoholic fatty liver disease. Fructose intake also causes features of metabolic syndrome in laboratory animals and humans. The first enzyme in fructose metabolism is fructokinase, which exists as two isoforms, A and C. Here we show that fructose-induced metabolic syndrome is prevented in mice lacking both isoforms but is exacerbated in mice lacking fructokinase A. Fructokinase C is expr...

  13. Differential Contributions of Nonmuscle Myosin II Isoforms and Functional Domains to Stress Fiber Mechanics

    OpenAIRE

    Ching-Wei Chang; Sanjay Kumar

    2015-01-01

    While is widely acknowledged that nonmuscle myosin II (NMMII) enables stress fibers (SFs) to generate traction forces against the extracellular matrix, little is known about how specific NMMII isoforms and functional domains contribute to SF mechanics. Here we combine biophotonic and genetic approaches to address these open questions. First, we suppress the NMMII isoforms MIIA and MIIB and apply femtosecond laser nanosurgery to ablate and investigate the viscoelastic retraction of individual ...

  14. Expression of mdr isoforms in mice during estrous cycle and under hormone stimulation

    OpenAIRE

    Marion Schiengold; Lavínia Schwantes; Ribeiro, Maria F; Nívia Lothhammer; Gonzalez, Tatiana P.; Jose Artur Bogo Chies; Nardi, Nance B

    2006-01-01

    The multidrug resistance (MDR) phenotype is associated with the expression of P-glycoprotein (Pgp), coded by the multigenic mdr family. Mice present the isoforms mdr1 and mdr3, which are responsible for multidrug resistance, and mdr2, that is involved in the transport of phospholipids. mdr1 expression has more recently been associated also with the secretion of steroid hormones. This work presents an RT-PCR analysis of the expression of mdr isoforms, in several organs of mice during different...

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

    OpenAIRE

    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. Distribution of tropomyosin isoforms in different types of single fibers isolated from bovine skeletal muscles.

    Science.gov (United States)

    Oe, M; Ojima, K; Nakajima, I; Chikuni, K; Shibata, M; Muroya, S

    2016-08-01

    To clarify the relationship between myosin heavy chain (MyHC) isoforms and tropomyosin (TPM) isoforms in single fibers, 64 single fibers were isolated from each of bovine three muscles (masseter, semispinalis and semitendinosus). mRNA expressions of MyHC and TPM isoforms were analyzed by real-time PCR. All single fibers from the masseter expressed MyHC-slow. The fibers from the semispinalis expressed both MyHC-slow and 2a. The fibers from the semitendinosus expressed MyHC-slow, 2a and 2x. TPM-1 and TPM-2 were co-expressed in 2a and 2x type fibers, and TPM-2 and TPM-3 were co-expressed in slow type fibers. The expression pattern of TPM isoforms in each fiber type was similar between fibers isolated from different muscles. These results suggest that TPM-1 and TPM-3 isoforms correspond to the function of 2a or 2x type fibers and slow type fibers, respectively, with TPM-2 in common. Furthermore, the patterns of MyHC and TPM isoform combinations did not vary among single fibers isolated from the individual muscles examined. PMID:27105153

  17. Two novel human NUMB isoforms provide a potential link between development and cancer

    Directory of Open Access Journals (Sweden)

    Prudovsky Igor

    2010-12-01

    Full Text Available Abstract We previously identified four functionally distinct human NUMB isoforms. Here, we report the identification of two additional isoforms and propose a link between the expression of these isoforms and cancer. These novel isoforms, NUMB5 and NUMB6, lack exon 10 and are expressed in cells known for polarity and migratory behavior, such as human amniotic fluid cells, glioblastoma and metastatic tumor cells. RT-PCR and luciferase assays demonstrate that NUMB5 and NUMB6 are less antagonistic to NOTCH signaling than other NUMB isoforms. Immunocytochemistry analyses show that NUMB5 and NUMB6 interact and complex with CDC42, vimentin and the CDC42 regulator IQGAP1 (IQ (motif GTPase activating protein 1. Furthermore, the ectopic expression of NUMB5 and NUMB6 induces the formation of lamellipodia (NUMB5 and filopodia (NUMB6 in a CDC42- and RAC1-dependent manner. These results are complemented by in vitro and in vivo studies, demonstrating that NUMB5 and NUMB6 alter the migratory behavior of cells. Together, these novel isoforms may play a role in further understanding the NUMB function in development and cancer.

  18. Profiling alternatively spliced mRNA isoforms for prostate cancer classification

    Directory of Open Access Journals (Sweden)

    Fan Jian-Bing

    2006-04-01

    Full Text Available Abstract Background Prostate cancer is one of the leading causes of cancer illness and death among men in the United States and world wide. There is an urgent need to discover good biomarkers for early clinical diagnosis and treatment. Previously, we developed an exon-junction microarray-based assay and profiled 1532 mRNA splice isoforms from 364 potential prostate cancer related genes in 38 prostate tissues. Here, we investigate the advantage of using splice isoforms, which couple transcriptional and splicing regulation, for cancer classification. Results As many as 464 splice isoforms from more than 200 genes are differentially regulated in tumors at a false discovery rate (FDR of 0.05. Remarkably, about 30% of genes have isoforms that are called significant but do not exhibit differential expression at the overall mRNA level. A support vector machine (SVM classifier trained on 128 signature isoforms can correctly predict 92% of the cases, which outperforms the classifier using overall mRNA abundance by about 5%. It is also observed that the classification performance can be improved using multivariate variable selection methods, which take correlation among variables into account. Conclusion These results demonstrate that profiling of splice isoforms is able to provide unique and important information which cannot be detected by conventional microarrays.

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

    Energy Technology Data Exchange (ETDEWEB)

    Ohlrogge, J.B.

    1989-01-01

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

  20. Characterization of 14-3-3 isoforms expressed in the Echinococcus granulosus pathogenic larval stage.

    Science.gov (United States)

    Teichmann, Aline; Vargas, Daiani M; Monteiro, Karina M; Meneghetti, Bruna V; Dutra, Cristine S; Paredes, Rodolfo; Galanti, Norbel; Zaha, Arnaldo; Ferreira, Henrique B

    2015-04-01

    The 14-3-3 protein family of eukaryotic regulators was studied in Echinococcus granulosus, the causative agent of cystic hydatid disease. These proteins mediate important cellular processes in eukaryotes and are expected to play important roles in parasite biology. Six isoforms of E. granulosus 14-3-3 genes and proteins (Eg14-3-3.1-6) were analyzed, and their phylogenetic relationships were established with bona fide 14-3-3 orthologous proteins from eukaryotic species. Eg14-3-3 isoforms with previous evidence of expression (Eg14-3-3.1-4) in E. granulosus pathogenic larval stage (metacestode) were cloned, and recombinant proteins were used for functional studies. These protein isoforms were detected in different components of E. granulosus metacestode, including interface components with the host. The roles that are played by Eg14-3-3 proteins in parasite biology were inferred from the repertoires of interacting proteins with each isoform, as assessed by gel overlay, cross-linking, and affinity chromatography assays. A total of 95 Eg14-3-3 protein ligands were identified by mass spectrometry. Eg14-3-3 isoforms have shared partners (44 proteins), indicating some overlapping functions; however, they also bind exclusive partners (51 proteins), suggesting Eg14-3-3 functional specialization. These ligand repertoires indicate the involvement of Eg14-3-3 proteins in multiple biochemical pathways in the E. granulosus metacestode and note some degree of isoform specialization.

  1. Distribution of tropomyosin isoforms in different types of single fibers isolated from bovine skeletal muscles.

    Science.gov (United States)

    Oe, M; Ojima, K; Nakajima, I; Chikuni, K; Shibata, M; Muroya, S

    2016-08-01

    To clarify the relationship between myosin heavy chain (MyHC) isoforms and tropomyosin (TPM) isoforms in single fibers, 64 single fibers were isolated from each of bovine three muscles (masseter, semispinalis and semitendinosus). mRNA expressions of MyHC and TPM isoforms were analyzed by real-time PCR. All single fibers from the masseter expressed MyHC-slow. The fibers from the semispinalis expressed both MyHC-slow and 2a. The fibers from the semitendinosus expressed MyHC-slow, 2a and 2x. TPM-1 and TPM-2 were co-expressed in 2a and 2x type fibers, and TPM-2 and TPM-3 were co-expressed in slow type fibers. The expression pattern of TPM isoforms in each fiber type was similar between fibers isolated from different muscles. These results suggest that TPM-1 and TPM-3 isoforms correspond to the function of 2a or 2x type fibers and slow type fibers, respectively, with TPM-2 in common. Furthermore, the patterns of MyHC and TPM isoform combinations did not vary among single fibers isolated from the individual muscles examined.

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

    Directory of Open Access Journals (Sweden)

    Valeria Hansberg-Pastor

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

  4. Expression of mdr isoforms in mice during estrous cycle and under hormone stimulation

    Directory of Open Access Journals (Sweden)

    Marion Schiengold

    2006-01-01

    Full Text Available The multidrug resistance (MDR phenotype is associated with the expression of P-glycoprotein (Pgp, coded by the multigenic mdr family. Mice present the isoforms mdr1 and mdr3, which are responsible for multidrug resistance, and mdr2, that is involved in the transport of phospholipids. mdr1 expression has more recently been associated also with the secretion of steroid hormones. This work presents an RT-PCR analysis of the expression of mdr isoforms, in several organs of mice during different phases of the estrous cycle. Additionally, females were ovariectomized, submitted to different hormone treatments, and their uterus was analyzed for the expression of mdr isoforms. The results show that in the adrenal gland and ovaries mdr1 is the main isoform during proestrus, and that progesterone or a combination of progesterone and estrogen induce the expression of all mdr isoforms in the uterus of ovariectomized females. We suggest that the functions of mdr1 and mdr3 are overlapping, that mdr3 may be the more efficient isoform in the detoxification function, and that mdr1 may be more closely related to the secretion of steroid hormones.

  5. Molecular characterization of human thyroid hormone receptor β isoform 4.

    Science.gov (United States)

    Moriyama, Kenji; Yamamoto, Hiroyuki; Futawaka, Kumi; Atake, Asami; Kasahara, Masato; Tagami, Tetsuya

    2016-01-01

    Thyroid hormone exerts a pleiotropic effect on development, differentiation, and metabolism through thyroid hormone receptor (TR). A novel thyroid hormone receptor β isoform (TRβ4) was cloned using PCR from a human pituitary cDNA library as a template. We report here the characterization of TRβ4 from a molecular basis. Temporal expression of TRβ4 during the fetal period is abundant in the brain and kidney, comparable with the adult pattern. Western blot analysis revealed that TRs are ubiquitination labile proteins, while TRβ1 is potentially stable. TRβ1, peroxisome proliferator-activated receptors (PPAR), and vitamin D receptor (VDR), which belong to class II transcription factors that function via the formation of heterodimeric complexes with retinoid X receptor (RXR), were suppressed by TRβ4 in a dose-dependent manner. Thus, TRβ4 exhibits ligand-independent transcriptional silencing, possibly as a substitute for dimerized RXR. In this study, TRβ1 and TRβ4 transcripts were detected in several cell lines. Quantitative RT-PCR assay showed that the expression of TRβ4 in human embryonic carcinoma cells of the testis was suppressed by sex hormone in a reciprocal manner to TRβ1. In contrast, TRβ4 was expressed under a high dose of triiodothyronine (T3) in a reciprocal manner to TRβ1. Finally, in transiently transfected NIH-3T3 cells, green fluorescence protein (GFP)-tagged TRβ4 was mostly nuclear in both the absence and the presence of T3. By mutating defined regions of both TRβs, we found that both TRβ1 and TRβ4 had altered nuclear/cytoplasmic distribution as compared with wild-type, and different to T3 and the nuclear receptor corepressor (NCoR). Thus, site-specific DNA binding is not essential for maintaining TRβs within the nucleus. PMID:26513165

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

    Directory of Open Access Journals (Sweden)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  8. The relationships among bovine αS-casein phosphorylation isoforms suggest different phosphorylation pathways.

    Science.gov (United States)

    Fang, Z H; Visker, M H P W; Miranda, G; Delacroix-Buchet, A; Bovenhuis, H; Martin, P

    2016-10-01

    Casein (CN) phosphorylation is an important posttranslational modification and is one of the key factors responsible for constructing and stabilizing casein micelles. Variation in phosphorylation degree of αS-CN is of great interest because it is suggested to affect milk technological properties. This study aimed to investigate the variation in phosphorylation degree of αS-CN among milk of individual cows and to explore relationships among different phosphorylation isoforms of αS-CN. For this purpose, we analyzed morning milk samples from 529 French Montbéliarde cows using liquid chromatography coupled with electrospray ionization mass spectrometry. We detected 3 new phosphorylation isoforms: αS2-CN-9P, αS2-CN-14P, and αS2-CN-15P in bovine milk, in addition to the known isoforms αS1-CN-8P, αS1-CN-9P, αS2-CN-10P, αS2-CN-11P, αS2-CN-12P, and αS2-CN-13P. The relative concentrations of each αS-CN phosphorylation isoform varied considerably among individual cows. Furthermore, the phenotypic correlations and hierarchical clustering suggest at least 2 regulatory systems for phosphorylation of αS-CN: one responsible for isoforms with lower levels of phosphorylation (αS1-CN-8P, αS2-CN-10P, and αS2-CN-11P), and another responsible for isoforms with higher levels of phosphorylation (αS1-CN-9P, αS2-CN-12P, αS2-CN-13P, and αS2-CN-14P). Identifying all phosphorylation sites of αS2-CN and investigating the genetic background of different αS2-CN phosphorylation isoforms may provide further insight into the phosphorylation mechanism of caseins. PMID:27522420

  9. Transferrin and Its Isoforms from Normal Human Serum Revealed by Several Analytical Techniques

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Transferrin(TF) and its isoforms have been widely reported via various analytical techniques, including a noticeable increased number of isoforms with low content of sialic acid(asialo-, monosialo-, and disialo-transferrin) and asialo-TF as well as disialo-TF, with one or several oligosaccharides released in human serum transferrin(hTf). Here,hTf has been purified by native gradient polyacrylamide gel electrophoresis(PAGENG) before use. The hTf extracted with the electron-transfer approach showed a single subunit band(77.1 Da) in the SDS-PAGE gel, but it exhibited two bands in the native and denatured isoelectric focusing(IEF) gels, namely, hTf-2Fe3+ and apo-hTf, without finding any other transferrin isoforms. A reversed phase HPLC(RP-HPLC) equipped with a C18 column effectively separated hTf and its polymers and combined off-line techniques, including peptide mass fingerprinting(PMF), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS) and database search, and identified the high homology among hTf, apo-hTf, and their isoforms. Moreover, the elution solution consisting of acetonitrile and formic acid could easily denature both hTf and apo-hTf to form various isoforms during separation with HPLC, indicating that chemical factors lead to the formation of various isoforms in transferrin, artificially, during extraction and separation.The authors claimed that only two transferrin isoforms existed in the NHS, namely, hTf-2Fe3+ and apo-hTf, which could be employed in biomarkers, to distinguish the healthy population from many disease sufferers, such as,carbohydrate-deficient transferrin(CDT).

  10. Differential CARM1 Isoform Expression in Subcellular Compartments and among Malignant and Benign Breast Tumors.

    Directory of Open Access Journals (Sweden)

    David Shlensky

    Full Text Available Coactivator-associated arginine methyltransferase 1 (CARM1 is a coactivator for ERα and cancer-relevant transcription factors, and can methylate diverse cellular targets including histones. CARM1 is expressed in one of two alternative splice isoforms, full-length CARM1 (CARM1FL and truncated CARM1 (CARM1ΔE15. CARM1FL and CARM1ΔE15 function differently in transcriptional regulation, protein methylation, and mediation of pre-mRNA splicing in cellular models.To investigate the functional roles and the prognosis potential of CARM1 alternative spliced isoforms in breast cancer, we used recently developed antibodies to detect differential CARM1 isoform expression in subcellular compartments and among malignant and benign breast tumors.Immunofluorescence in MDA-MB-231 and BG-1 cell lines demonstrated that CARM1ΔE15 is the dominant isoform expressed in the cytoplasm, and CARM1FL is more nuclear localized. CARM1ΔE15 was found to be more sensitive to Hsp90 inhibition than CARM1FL, indicating that the truncated isoform may be the oncogenic form. Clinical cancer samples did not have significantly higher expression of CARM1FL or CARM1ΔE15 than benign breast samples at the level of mRNA or histology. Furthermore neither CARM1FL nor CARM1ΔE15 expression correlated with breast cancer molecular subtypes, tumor size, or lymph node involvement.The analysis presented here lends new insights into the possible oncogenic role of CARM1ΔE15. This study also demonstrates no obvious association of CARM1 isoform expression and clinical correlates in breast cancer. Recent studies, however, have shown that CARM1 expression correlates with poor prognosis, indicating a need for further studies of both CARM1 isoforms in a large cohort of breast cancer specimens.

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

    Science.gov (United States)

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

    2004-01-01

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

  12. Differential dynamics of RAS isoforms in GDP- and GTP-bound states.

    Science.gov (United States)

    Kapoor, Abhijeet; Travesset, Alex

    2015-06-01

    RAS subfamily proteins regulates cell growth promoting signaling processes by cycling between active (GTP-bound) and inactive (GDP-bound) states. Different RAS isoforms, though structurally similar, exhibit functional specificity and are associated with different types of cancers and developmental disorders. Understanding the dynamical differences between the isoforms is crucial for the design of inhibitors that can selectively target a particular malfunctioning isoform. In this study, we provide a comprehensive comparison of the dynamics of all the three RAS isoforms (HRAS, KRAS, and NRAS) using extensive molecular dynamics simulations in both the GDP- (total of 3.06 μs) and GTP-bound (total of 2.4 μs) states. We observed significant differences in the dynamics of the isoforms, which rather interestingly, varied depending on the type of the nucleotide bound and the simulation temperature. Both SwitchI (Residues 25-40) and SwitchII (Residues 59-75) differ significantly in their flexibility in the three isoforms. Furthermore, Principal Component Analysis showed that there are differences in the conformational space sampled by the GTP-bound RAS isoforms. We also identified a previously unreported pocket, which opens transiently during MD simulations, and can be targeted to regulate nucleotide exchange reaction or possibly interfere with membrane localization. Further, we present the first simulation study showing GDP destabilization in the wild-type RAS protein. The destabilization of GDP/GTP occurred only in 1/50 simulations, emphasizing the need of guanine nucleotide exchange factors (GEFs) to accelerate such an extremely unfavorable process. This observation along with the other results presented in this article further support our previously hypothesized mechanism of GEF-assisted nucleotide exchange.

  13. Two isoforms of trimming glucosidase II exist in mammalian tissues and cell lines but not in yeast and insect cells.

    Science.gov (United States)

    Ziak, M; Meier, M; Etter, K S; Roth, J

    2001-01-12

    We previously cloned glucosidase II and provided in vivo evidence for its involvement in protein folding quality control. DNA-sequencing of different clones demonstrated the existence of two isoforms of glucosidase II which differed by 66 nucleotides due to alternative splicing. The existence of two enzyme isoforms in various organs of pig and rat as well as human, bovine, rat, and mouse cell lines could be demonstrated by RT-PCR and Western blotting. Furthermore, the two isoforms of glucosidase II could be detected in embryonic and postnatal rat kidney and liver. In yeast, Saccharomyces cerevisiae, and in insects, Drosophila S2 cells, only one isoforms of the enzyme was detectable. The ubiquitous occurrence of the two glucosidase II isoforms in mammalian tissues and cell lines might be indicative of a special function of each isoform.

  14. Dichotomy of short and long thymic stromal lymphopoietin isoforms in inflammatory disorders of the bowel and skin

    Science.gov (United States)

    Fornasa, Giulia; Tsilingiri, Katerina; Caprioli, Flavio; Botti, Fiorenzo; Mapelli, Marina; Meller, Stephan; Kislat, Andreas; Homey, Bernhard; Di Sabatino, Antonio; Sonzogni, Angelica; Viale, Giuseppe; Diaferia, Giuseppe; Gori, Alessandro; Longhi, Renato; Penna, Giuseppe; Rescigno, Maria

    2015-01-01

    Background Thymic stromal lymphopoietin (TSLP) is a cytokine with pleiotropic functions in the immune system. It has been associated with allergic reactions in the skin and lungs but also homeostatic tolerogenic responses in the thymus and gut. Objective In human subjects TSLP is present in 2 isoforms, short and long. Here we wanted to investigate the differential expression of the TSLP isoforms and discern their biological implications under homeostatic or inflammatory conditions. Methods We evaluated the expression of TSLPs in tissues from healthy subjects, patients with ulcerative colitis, patients with celiac disease, and patients with atopic dermatitis and on epithelial cells and keratinocytes under steady-state conditions or after stimulation. We then tested the immune activity of TSLP isoforms both in vitro and in vivo. Results We showed that TSLP isoforms are responsible for 2 opposite immune functions. The short isoform is expressed under steady-state conditions and exerts anti-inflammatory activities by affecting the capacity of PBMCs and dendritic cells to produce inflammatory cytokines. Moreover, the short isoform TSLP ameliorates experimental colitis in mice and prevents endotoxin shock. The long isoform of TSLP is proinflammatory and is only expressed during inflammation. The isoforms are differentially regulated by pathogenic bacteria, such as Salmonella species and adhesive-invasive Escherichia coli. Conclusions We have solved the dilemma of TSLP being both homeostatic and inflammatory. The TSLP isoform ratio is altered during several inflammatory disorders, with strong implications in disease treatment and prevention. Indeed, targeting of the long isoform of TSLP at the C-terminal portion, which is common to both isoforms, might lead to unwanted side effects caused by neutralization of the homeostatic short isoform. PMID:26014813

  15. Computational Estimates of Binding Affinities for Estrogen Receptor Isoforms in Rainbow Trout

    CERN Document Server

    Shyu, Conrad

    2009-01-01

    Molecular dynamics simulations are performed to determine the binding affinities between the hormone 17 beta-estradiol (E2) and different estrogen receptor (ER) isoforms in the rainbow trout (Oncorhynchus mykiss). Previous studies have demonstrated that a recent, unique gene duplication of the ER alpha subtype created two isoforms ER alpha 1 and ER alpha 2, and an early secondary split of ER beta produced two distinct isoforms of ER beta 1 and ER beta 2 based on the phylogenetic analysis. The objective of our computational studies is to provide insight into the underlying evolutionary selection pressure on the ER isoforms. Our results show that E2 binds preferentially to ER alpha 1. This finding corresponds to the experimental results as the ERs evolved from gene duplication events are frequently free from selective pressure and should exhibit no deleterious effects. The E2, however, only binds slightly better to ER beta 2. Both isoforms remain competitive. This finding reflects the fact that since ER beta 2 ...

  16. Silencing GFAP isoforms in astrocytoma cells disturbs laminin-dependent motility and cell adhesion.

    Science.gov (United States)

    Moeton, Martina; Kanski, Regina; Stassen, Oscar M J A; Sluijs, Jacqueline A; Geerts, Dirk; van Tijn, Paula; Wiche, Gerhard; van Strien, Miriam E; Hol, Elly M

    2014-07-01

    Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed in astrocytes and neural stem cells. The GFAP gene is alternatively spliced, and expression of GFAP is highly regulated during development, on brain damage, and in neurodegenerative diseases. GFAPα is the canonical splice variant and is expressed in all GFAP-positive cells. In the human brain, the alternatively spliced transcript GFAPδ marks specialized astrocyte populations, such as subpial astrocytes and the neurogenic astrocytes in the human subventricular zone. We here show that shifting the GFAP isoform ratio in favor of GFAPδ in astrocytoma cells, by selectively silencing the canonical isoform GFAPα with short hairpin RNAs, induced a change in integrins, a decrease in plectin, and an increase in expression of the extracellular matrix component laminin. Together, this did not affect cell proliferation but resulted in a significantly decreased motility of astrocytoma cells. In contrast, a down-regulation of all GFAP isoforms led to less cell spreading, increased integrin expression, and a >100-fold difference in the adhesion of astrocytoma cells to laminin. In summary, isoform-specific silencing of GFAP revealed distinct roles of a specialized GFAP network in regulating the interaction of astrocytoma cells with the extracellular matrix through laminin.-Moeton, M., Kanski, R., Stassen, O. M. J. A., Sluijs, J. A., Geerts, D., van Tijn, P., Wiche, G., van Strien, M. E., Hol, E. M. Silencing GFAP isoforms in astrocytoma cells disturbs laminin dependent motility and cell adhesion.

  17. iReckon: simultaneous isoform discovery and abundance estimation from RNA-seq data.

    Science.gov (United States)

    Mezlini, Aziz M; Smith, Eric J M; Fiume, Marc; Buske, Orion; Savich, Gleb L; Shah, Sohrab; Aparicio, Sam; Chiang, Derek Y; Goldenberg, Anna; Brudno, Michael

    2013-03-01

    High-throughput RNA sequencing (RNA-seq) promises to revolutionize our understanding of genes and their role in human disease by characterizing the RNA content of tissues and cells. The realization of this promise, however, is conditional on the development of effective computational methods for the identification and quantification of transcripts from incomplete and noisy data. In this article, we introduce iReckon, a method for simultaneous determination of the isoforms and estimation of their abundances. Our probabilistic approach incorporates multiple biological and technical phenomena, including novel isoforms, intron retention, unspliced pre-mRNA, PCR amplification biases, and multimapped reads. iReckon utilizes regularized expectation-maximization to accurately estimate the abundances of known and novel isoforms. Our results on simulated and real data demonstrate a superior ability to discover novel isoforms with a significantly reduced number of false-positive predictions, and our abundance accuracy prediction outmatches that of other state-of-the-art tools. Furthermore, we have applied iReckon to two cancer transcriptome data sets, a triple-negative breast cancer patient sample and the MCF7 breast cancer cell line, and show that iReckon is able to reconstruct the complex splicing changes that were not previously identified. QT-PCR validations of the isoforms detected in the MCF7 cell line confirmed all of iReckon's predictions and also showed strong agreement (r(2) = 0.94) with the predicted abundances. PMID:23204306

  18. Electrophoretic Mobility of Cardiac Myosin Heavy Chain Isoforms Revisited: Application of MALDI TOF/TOF Analysis

    Directory of Open Access Journals (Sweden)

    Petra Arnostova

    2011-01-01

    Full Text Available The expression of two cardiac myosin heavy chain (MyHC isoforms in response to the thyroid status was studied in left ventricles (LVs of Lewis rats. Major MyHC isoform in euthyroid and hyperthyroid LVs had a higher mobility on SDS-PAGE, whereas hypothyroid LVs predominantly contained a MyHC isoform with a lower mobility corresponding to that of the control soleus muscle. By comparing the MyHC profiles obtained under altered thyroid states together with the control soleus, we concluded that MyHCα was represented by the lower band with higher mobility and MyHCβ by the upper band. The identity of these two bands in SDS-PAGE gels was confirmed by western blot and mass spectrometry. Thus, in contrast to the literature data, we found that the MyHCα possessed a higher mobility rate than the MyHCβ isoform. Our data highlighted the importance of the careful identification of the MyHCα and MyHCβ isoforms analyzed by the SDS-PAGE.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

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

    2015-06-26

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

  1. Nesprin-2 epsilon: A novel nesprin isoform expressed in human ovary and Ntera-2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Lam, Le Thanh [Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry SY10 7AG (United Kingdom); Boehm, Sabrina V.; Roberts, Roland G. [Department of Medical and Molecular Genetics, King' s College London, London SE1 9RT (United Kingdom); Morris, Glenn E., E-mail: glennmanc@hotmail.com [Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry SY10 7AG (United Kingdom); Institute of Science and Technology in Medicine, Keele University, Keele ST5 5BG (United Kingdom)

    2011-08-26

    Highlights: {yields} A novel epsilon isoform of nesprin-2 has been discovered. {yields} This 120 kDa protein was predicted by bioinformatic analysis, but has not previously been observed. {yields} It is the main isoform expressed in a teratocarcinoma cell line and is also found in ovary. {yields} Like other nesprins, it is located at the nuclear envelope. {yields} We suggest it may have a role in very early development or in some ovary-specific function. -- Abstract: The nuclear envelope-associated cytoskeletal protein, nesprin-2, is encoded by a large gene containing several internal promoters that produce shorter isoforms. In a study of Ntera-2 teratocarcinoma cells, a novel isoform, nesprin-2-epsilon, was found to be the major mRNA and protein product of the nesprin-2 gene. Its existence was predicted by bioinformatic analysis, but this is the first direct demonstration of both the mRNA and the 120 kDa protein which is located at the nuclear envelope. In a panel of 21 adult and foetal human tissues, the nesprin-2-epsilon mRNA was strongly expressed in ovary but was a minor isoform elsewhere. The expression pattern suggests a possible link with very early development and a likely physiological role in ovary.

  2. Alterations of Lymphoid Enhancer Factor-1 Isoform Expression in Solid Tumors and Acute Leukemias

    Institute of Scientific and Technical Information of China (English)

    Wenbing WANG; Carsten M(U)LLER-TIDOW; Ping JI; Bj(o)rn STEFFEN; Ralf METZGER; Paul M. SCHNEIDER; Hartmut HALFTER; Mark SCHRADER; Wolfgang E. BERDEL; Hubert SERVE

    2005-01-01

    Two major transcripts of lymphoid enhancer factor-1 (LEF-1) have been described. The long isoform with β-catenin binding domain functions as a transcriptional enhancer factor. The short isoform derives from an intronic promoter and exhibits dominant negative activity. Recently, alterations of LEF- 1isoforms distribution have been described in colon cancer. In the current study we employed a quantitative real-time reverse transcription PCR method (TaqMan) to analyze expression of LEF-1 isoforms in a large cohort of human tumor (n=304) and tumor-free control samples (n=56). The highest expression level of LEF-1 was found in carcinoma samples whereas brain cancer samples expressed little. Expression of LEF1 was different in distinct cancer types. For example, the mRNA level of LEF-1 was lower in testicular tumor samples compared with tumor-free control samples. Besides epithelial cancers, significant LEF-1expression was also found in hematopoietic cells. In hematological malignancies, overall LEF-1 level was higher in lymphocytic leukemias compared with myeloid leukemias and normal hematopoiesis. However,acute myeloid leukemia and acute lymphocytic leukemia showed a significantly increased fraction of the oncogenic LEF-1 compared with chronic lymphocytic leukemia and chronic myeloid leukemia. Taken together,these data suggest that LEF-1 is abundantly expressed in human tumors and the ratio of the oncogenic and the dominant negative short isoform altered not only in carcinomas but also in leukemia.

  3. Two isoforms of aquaporin 2 responsive to hypertonic stress in the bottlenose dolphin.

    Science.gov (United States)

    Suzuki, Miwa; Wakui, Hitomi; Itou, Takuya; Segawa, Takao; Inoshima, Yasuo; Maeda, Ken; Kikuchi, Kiyoshi

    2016-04-15

    This study investigated the expression of aquaporin 2 (AQP2) and its newly found alternatively spliced isoform (alternative AQP2) and the functions of these AQP2 isoforms in the cellular hyperosmotic tolerance in the bottlenose dolphin, ITALIC! Tursiops truncatus mRNA sequencing revealed that alternative AQP2 lacks the fourth exon and instead has a longer third exon that includes a part of the original third intron. The portion of the third intron, now part of the coding region of alternative AQP2, is highly conserved among many species of the order Cetacea but not among terrestrial mammals. Semi-quantitative PCR revealed that AQP2 was expressed only in the kidney, similar to terrestrial mammals. In contrast, alternative AQP2 was expressed in all organs examined, with strong expression in the kidney. In cultured renal cells, expression of both AQP2 isoforms was upregulated by the addition to the medium of NaCl but not by the addition of mannitol, indicating that the expression of both isoforms is induced by hypersalinity. Treatment with small interfering RNA for both isoforms resulted in a decrease in cell viability in hypertonic medium (500 mOsm kg(-1)) when compared with controls. These findings indicate that the expression of alternatively spliced AQP2 is ubiquitous in cetacean species, and it may be one of the molecules important for cellular osmotic tolerance throughout the body. PMID:26944501

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

    Science.gov (United States)

    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

  5. Serum amyloid A isoforms in serum and synovial fluid from spontaneously diseased dogs with joint diseases or other conditions

    DEFF Research Database (Denmark)

    Kjelgaard-Hansen, Mads Jens; Christensen, Michelle B.; Lee, Marcel Huisung;

    2007-01-01

    in samples obtained from dogs (n = 16) suffering from different inflammatory or non-inflammatory conditions, which were either related or unrelated to joints. Expression of SAA isoforms was visualized by denaturing isoelectric focusing and Western blotting. Serum amyloid A was present in serum from all dogs...... with systemic inflammatory activity, and up to four major isoforms with apparent isoelectric points between 6.1 and 7.9 were identified. In synovial fluid from inflamed joints one or more highly alkaline SAA isoforms (with apparent isoelectric points above 9.3) were identified, with data suggesting local...... production of these isoforms in the canine inflamed joint....

  6. Aryl hydrocarbon receptor nuclear translocator (ARNT) isoforms control lymphoid cancer cell proliferation through differentially regulating tumor suppressor p53 activity.

    Science.gov (United States)

    Gardella, Kacie A; Muro, Israel; Fang, Gloria; Sarkar, Krishnakali; Mendez, Omayra; Wright, Casey W

    2016-03-01

    The aryl hydrocarbon receptor nuclear translocator (ARNT) is involved in xenobiotic and hypoxic responses, and we previously showed that ARNT also regulates nuclear factor-κB (NF-κB) signaling by altering the DNA binding activity of the RelB subunit. However, our initial study of ARNT-mediated RelB modulation was based on simultaneous suppression of the two ARNT isoforms, isoform 1 and 3, and precluded the examination of their individual functions. We find here that while normal lymphocytes harbor equal levels of isoform 1 and 3, lymphoid malignancies exhibit a shift to higher levels of ARNT isoform 1. These elevated levels of ARNT isoform 1 are critical to the proliferation of these cancerous cells, as suppression of isoform 1 in a human multiple myeloma (MM) cell line, and an anaplastic large cell lymphoma (ALCL) cell line, triggered S-phase cell cycle arrest, spontaneous apoptosis, and sensitized cells to doxorubicin treatment. Furthermore, co-suppression of RelB or p53 with ARNT isoform 1 prevented cell cycle arrest and blocked doxorubicin induced apoptosis. Together our findings reveal that certain blood cancers rely on ARNT isoform 1 to potentiate proliferation by antagonizing RelB and p53-dependent cell cycle arrest and apoptosis. Significantly, our results identify ARNT isoform 1 as a potential target for anticancer therapies.

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

    OpenAIRE

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

    2013-01-01

    Mature, but not immature, dendritic cells are sensitive to glucocorticoid-induced apoptosis.Mature, but not immature, dendritic cells express proapoptotic glucocorticoid receptor translational isoforms.

  8. Exposing the specific roles of the invariant chain isoforms in shaping the MHC class II peptidome

    Directory of Open Access Journals (Sweden)

    Jean-Simon eFortin

    2013-12-01

    Full Text Available The peptide repertoire (peptidome associated with MHC class II molecules (MHCIIs is influenced by the polymorphic nature of the peptide binding groove but also by cell-intrinsic factors. The invariant chain (Ii chaperones MHCIIs, affecting their folding and trafficking. Recent discoveries relating to Ii functions have provided insights as to how it edits the MHCII peptidome. In humans, the Ii gene encodes four different isoforms for which structure-function analyses have highlighted common properties but also some non-redundant roles. Another layer of complexity arises from the fact that Ii heterotrimerizes, a characteristic that has the potential to affect the maturation of associated MHCIIs in many different ways, depending on the isoform combinations. Here, we emphasize the peptide editing properties of Ii and discuss the impact of the various isoforms on the MHCII peptidome.

  9. Epidermal growth-factor-induced transcript isoform variation drives mammary cell migration.

    Directory of Open Access Journals (Sweden)

    Wolfgang J Köstler

    Full Text Available Signal-induced transcript isoform variation (TIV includes alternative promoter usage as well as alternative splicing and alternative polyadenylation of mRNA. To assess the phenotypic relevance of signal-induced TIV, we employed exon arrays and breast epithelial cells, which migrate in response to the epidermal growth factor (EGF. We show that EGF rapidly--within one hour--induces widespread TIV in a significant fraction of the transcriptome. Importantly, TIV characterizes many genes that display no differential expression upon stimulus. In addition, similar EGF-dependent changes are shared by a panel of mammary cell lines. A functional screen, which utilized isoform-specific siRNA oligonucleotides, indicated that several isoforms play essential, non-redundant roles in EGF-induced mammary cell migration. Taken together, our findings highlight the importance of TIV in the rapid evolvement of a phenotypic response to extracellular signals.

  10. Alternative RNA splicing of KSHV ORF57 produces two different RNA isoforms.

    Science.gov (United States)

    Majerciak, Vladimir; Zheng, Zhi-Ming

    2016-01-15

    In lytically infected B cells Kaposi sarcoma-associated herpesvirus (KSHV) ORF57 gene encodes two RNA isoforms by alternative splicing of its pre-mRNA, which contains a small, constitutive intron in its 5' half and a large, suboptimal intron in its 3's half. The RNA1 isoform encodes full-length ORF57 and is a major isoform derived from splicing of the constitutive small intron, but retaining the suboptimal large intron as the coding region. A small fraction (splicing to produce a smaller non-coding RNA2 due to lack of a translational termination codon. Both RNAs are cleaved and polyadenylated at the same cleavage site CS83636. The insertion of ORF57 RNA1 into a restriction cutting site in certain mammalian expression vectors activates splicing of the subopitmal intron and produces a truncated ORF57 protein.

  11. Structural and Functional Characterization of Recombinant Isoforms of the Lentil Lipid Transfer Protein.

    Science.gov (United States)

    Bogdanov, I V; Finkina, E I; Balandin, S V; Melnikova, D N; Stukacheva, E A; Ovchinnikova, T V

    2015-01-01

    The recombinant isoforms Lc-LTP1 and Lc-LTP3 of the lentil lipid transfer protein were overexpressed in E. coli cells. It was confirmed that both proteins are stabilized by four disulfide bonds and characterized by a high proportion of the α-helical structure. It was found that Lc-LTP1 and Lc-LTP3 possess antimicrobial activity and can bind fatty acids. Both isoforms have the ability to bind specific IgE from sera of patients with food allergies, which recognize similar epitopes of the major peach allergen Pru p 3. Both isoforms were shown to have immunological properties similar to those of other plant allergenic LTPs, but Lc-LTP3 displayed a less pronounced immunoreactivity. PMID:26483961

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    Together with Parkinson's disease (PD) and dementia with Lewy bodies (DLB), Multiple System Atrophy (MSA) is a member of a diverse group of neurodegenerative disorders termed α-synucleinopathies. Previously, it has been shown that alpha-synuclein, parkin and synphilin-1 display disease specific...... controls using isoform-specific primers and exon specific antibodies in substantia nigra, striatum, cerebellar cortex, and nucleus dentatus. These regions are severely affected by alpha-synuclein pathology and neurodegeneration. Further, we have also investigated transcript levels for parkin and synphilin...... 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...

  13. The respiratory chain supercomplex organization is independent of COX7a2l isoforms.

    Science.gov (United States)

    Mourier, Arnaud; Matic, Stanka; Ruzzenente, Benedetta; Larsson, Nils-Göran; Milenkovic, Dusanka

    2014-12-01

    The organization of individual respiratory chain complexes into supercomplexes or respirasomes has attracted great interest because of the implications for cellular energy conversion. Recently, it was reported that commonly used mouse strains harbor a short COX7a2l (SCAFI) gene isoform that supposedly precludes the formation of complex IV-containing supercomplexes. This claim potentially has serious implications for numerous mouse studies addressing important topics in metabolism, including adaptation to space flights. Using several complementary experimental approaches, we show that mice with the short COX7a2l isoform have normal biogenesis and steady-state levels of complex IV-containing supercomplexes and consequently have normal respiratory chain function. Furthermore, we use a mouse knockout of Lrpprc and show that loss of complex IV compromises respirasome formation. We conclude that the presence of the short COX7a2l isoform in the commonly used C57BL/6 mouse strains does not prevent their use in metabolism research.

  14. Protein interaction network of alternatively spliced isoforms from brain links genetic risk factors for autism.

    Science.gov (United States)

    Corominas, Roser; Yang, Xinping; Lin, Guan Ning; Kang, Shuli; Shen, Yun; Ghamsari, Lila; Broly, Martin; Rodriguez, Maria; Tam, Stanley; Trigg, Shelly A; Fan, Changyu; Yi, Song; Tasan, Murat; Lemmens, Irma; Kuang, Xingyan; Zhao, Nan; Malhotra, Dheeraj; Michaelson, Jacob J; Vacic, Vladimir; Calderwood, Michael A; Roth, Frederick P; Tavernier, Jan; Horvath, Steve; Salehi-Ashtiani, Kourosh; Korkin, Dmitry; Sebat, Jonathan; Hill, David E; Hao, Tong; Vidal, Marc; Iakoucheva, Lilia M

    2014-04-11

    Increased risk for autism spectrum disorders (ASD) is attributed to hundreds of genetic loci. The convergence of ASD variants have been investigated using various approaches, including protein interactions extracted from the published literature. However, these datasets are frequently incomplete, carry biases and are limited to interactions of a single splicing isoform, which may not be expressed in the disease-relevant tissue. Here we introduce a new interactome mapping approach by experimentally identifying interactions between brain-expressed alternatively spliced variants of ASD risk factors. The Autism Spliceform Interaction Network reveals that almost half of the detected interactions and about 30% of the newly identified interacting partners represent contribution from splicing variants, emphasizing the importance of isoform networks. Isoform interactions greatly contribute to establishing direct physical connections between proteins from the de novo autism CNVs. Our findings demonstrate the critical role of spliceform networks for translating genetic knowledge into a better understanding of human diseases.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  19. In vivo human apolipoprotein E isoform fractional turnover rates in the CNS.

    Directory of Open Access Journals (Sweden)

    Kristin R Wildsmith

    Full Text Available Apolipoprotein E (ApoE is the strongest genetic risk factor for Alzheimer's disease and has been implicated in the risk for other neurological disorders. The three common ApoE isoforms (ApoE2, E3, and E4 each differ by a single amino acid, with ApoE4 increasing and ApoE2 decreasing the risk of Alzheimer's disease (AD. Both the isoform and amount of ApoE in the brain modulate AD pathology by altering the extent of amyloid beta (Aβ peptide deposition. Therefore, quantifying ApoE isoform production and clearance rates may advance our understanding of the role of ApoE in health and disease. To measure the kinetics of ApoE in the central nervous system (CNS, we applied in vivo stable isotope labeling to quantify the fractional turnover rates of ApoE isoforms in 18 cognitively-normal adults and in ApoE3 and ApoE4 targeted-replacement mice. No isoform-specific differences in CNS ApoE3 and ApoE4 turnover rates were observed when measured in human CSF or mouse brain. However, CNS and peripheral ApoE isoform turnover rates differed substantially, which is consistent with previous reports and suggests that the pathways responsible for ApoE metabolism are different in the CNS and the periphery. We also demonstrate a slower turnover rate for CSF ApoE than that for amyloid beta, another molecule critically important in AD pathogenesis.

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

    Directory of Open Access Journals (Sweden)

    Tanja Seeger

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

  1. Crystal structures of a halophilic archaeal malate synthase from Haloferax volcanii and comparisons with isoforms A and G

    Directory of Open Access Journals (Sweden)

    Thomas Geoffrey C

    2011-05-01

    Full Text Available Abstract Background Malate synthase, one of the two enzymes unique to the glyoxylate cycle, is found in all three domains of life, and is crucial to the utilization of two-carbon compounds for net biosynthetic pathways such as gluconeogenesis. In addition to the main isoforms A and G, so named because of their differential expression in E. coli grown on either acetate or glycolate respectively, a third distinct isoform has been identified. These three isoforms differ considerably in size and sequence conservation. The A isoform (MSA comprises ~530 residues, the G isoform (MSG is ~730 residues, and this third isoform (MSH-halophilic is ~430 residues in length. Both isoforms A and G have been structurally characterized in detail, but no structures have been reported for the H isoform which has been found thus far only in members of the halophilic Archaea. Results We have solved the structure of a malate synthase H (MSH isoform member from Haloferax volcanii in complex with glyoxylate at 2.51 Å resolution, and also as a ternary complex with acetyl-coenzyme A and pyruvate at 1.95 Å. Like the A and G isoforms, MSH is based on a β8/α8 (TIM barrel. Unlike previously solved malate synthase structures which are all monomeric, this enzyme is found in the native state as a trimer/hexamer equilibrium. Compared to isoforms A and G, MSH displays deletion of an N-terminal domain and a smaller deletion at the C-terminus. The MSH active site is closely superimposable with those of MSA and MSG, with the ternary complex indicating a nucleophilic attack on pyruvate by the enolate intermediate of acetyl-coenzyme A. Conclusions The reported structures of MSH from Haloferax volcanii allow a detailed analysis and comparison with previously solved structures of isoforms A and G. These structural comparisons provide insight into evolutionary relationships among these isoforms, and also indicate that despite the size and sequence variation, and the truncated C

  2. Involvement of specific calmodulin isoforms in salicylic acid-independent activation of plant disease resistance responses

    OpenAIRE

    Heo, Won Do; Lee, Sang Hyoung; Kim, Min Chul; Kim, Jong Cheol; Chung, Woo Sik; Chun, Hyun Jin; Lee, Kyoung Joo; Park, Chan Young; Park, Hyeong Cheol; Choi, Ji Young; Cho, Moo Je

    1999-01-01

    The Ca2+ signal is essential for the activation of plant defense responses, but downstream components of the signaling pathway are still poorly defined. Here we demonstrate that specific calmodulin (CaM) isoforms are activated by infection or pathogen-derived elicitors and participate in Ca2+-mediated induction of plant disease resistance responses. Soybean CaM (SCaM)-4 and SCaM-5 genes, which encode for divergent CaM isoforms, were induced within 30 min by a fungal elicitor or pathogen, wher...

  3. Two farnesoid X receptor alpha isoforms in Japanese medaka (Oryzias latipes) are differentially activated in vitro

    OpenAIRE

    Howarth, Deanna L.; Hagey, Lee R.; Law, Sheran H.W.; Ai, Ni; Krasowski, Matthew D.; Ekins, Sean; Moore, John T.; Erin M Kollitz; Hinton, David E.; Kullman, Seth W.

    2010-01-01

    The nuclear receptor farnesoid X receptor alpha (FXRα, NR1H4) is activated by bile acids in multiple species including mouse, rat, and human and in this study we have identified two isoforms of Fxrα in Japanese medaka (Oryzias latipes), a small freshwater teleost. Both isoforms share a high amino acid sequence identity to mammalian FXRα (~70% in the ligand-binding domain). Fxrα1 and Fxrα2 differ within the AF1 domain due to alternative splicing at the fourth intron-exon boundary. This process...

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

    DEFF Research Database (Denmark)

    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...

  5. Each Individual Isoform of the Dopamine D2 Receptor Protects from Lactotroph Hyperplasia

    OpenAIRE

    Radl, Daniela; De Mei, Claudia; Chen, Eric; Lee, Hyuna; Borrelli, Emiliana

    2013-01-01

    Dopamine acting through D2 receptors (D2Rs) controls lactotroph proliferation and prolactin (PRL) levels. Ablation of this receptor in mice results in lactotroph hyperplasia and prolactinomas in aged females. Alternative splicing of the Drd2 gene generates 2 independent isoforms, a long (D2L) and a short (D2S) isoform, which are present in all D2R-expressing cells. Here, we addressed the role of D2L and D2S on lactotroph physiology through the generation and analysis of D2S-null mice and thei...

  6. Role of Sec24 isoforms in selective export of membrane proteins from the endoplasmic reticulum

    OpenAIRE

    Wendeler, Markus W; Paccaud, Jean-Pierre; Hauri, Hans-Peter

    2007-01-01

    Sec24 of the COPII (coat protein complex II) vesicle coat mediates the selective export of membrane proteins from the endoplasmic reticulum (ER) in yeast. Human cells express four Sec24 isoforms, but their role is unknown. Here, we report the differential effects of Sec24 isoform-specific silencing on the transport of the membrane reporter protein ERGIC-53 (ER–Golgi intermediate compartment-53) carrying the cytosolic ER export signals di-phenylalanine, di-tyrosine, di-leucine, di-isoleucine, ...

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

    OpenAIRE

    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 ...

  8. Distribution of protein kinase Mzeta and the complete protein kinase C isoform family in rat brain

    DEFF Research Database (Denmark)

    Naik, M U; Benedikz, Eirikur; Hernandez, I;

    2000-01-01

    Protein kinase C (PKC) is a multigene family of at least ten isoforms, nine of which are expressed in brain (alpha, betaI, betaII, gamma, delta, straightepsilon, eta, zeta, iota/lambda). Our previous studies have shown that many of these PKCs participate in synaptic plasticity in the CA1 region of......, protein kinase Mzeta (PKMzeta). In this study, we used immunoblot and immunocytochemical techniques with isoform-specific antisera to examine the distribution of the complete family of PKC isozymes and PKMzeta in rat brain. Each form of PKC showed a widespread distribution in the brain with a distinct...

  9. Structural and Functional Characterization of Recombinant Isoforms of the Lentil Lipid Transfer Protein

    OpenAIRE

    Bogdanov, I. V.; Finkina, E. I.; Balandin, S. V.; Melnikova, D. N.; Stukacheva, E. A.; Ovchinnikova, T. V.

    2015-01-01

    The recombinant isoforms Lc-LTP1 and Lc-LTP3 of the lentil lipid transfer protein were overexpressed in E. coli cells. It was confirmed that both proteins are stabilized by four disulfide bonds and characterized by a high proportion of the α-helical structure. It was found that Lc-LTP1 and Lc-LTP3 possess antimicrobial activity and can bind fatty acids. Both isoforms have the ability to bind specific IgE from sera of patients with food allergies, which recognize similar epitopes of the major ...

  10. STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF RECOMBINANT ISOFORMS OF THE LENTIL LIPID TRANSFER PROTEIN

    OpenAIRE

    Bogdanov, I. V.; Finkina, E. I.; Balandin, S. V.; Melnikova, D. N.; Stukacheva, E. A.; Ovchinnikova, T. V.

    2015-01-01

    The recombinant isoforms Lc-LTP1 and Lc-LTP3 of the lentil lipid transfer protein were overexpressed in E. coli cells. It was confirmed that both proteins are stabilized by four disulfide bonds and characterized by a high proportion of the α-helical structure. It was found that Lc-LTP1 and Lc-LTP3 possess antimicrobial activity and can bind fatty acids. Both isoforms have the ability to bind specific IgE from sera of patients with food allergies, which recognize similar epitopes of the major ...

  11. Lipoprotein(a) concentrations, isoform size, and risk of type 2 diabetes

    DEFF Research Database (Denmark)

    Kamstrup, Pia Rørbæk; Nordestgaard, Børge

    2013-01-01

    study to investigate whether large isoform size, low concentrations in plasma, or both, are causally associated with type 2 diabetes. METHODS: We assessed data for adults from the Danish general population enrolled in the Copenhagen City Heart Study and the Copenhagen General Population Study, with and......(a) concentrations alone seem not to be causally associated with type 2 diabetes, but a causal association for large lipoprotein(a) isoform size cannot be excluded. FUNDING: Danish Heart Foundation, Danish Council for Independent Research-Medical Sciences, IMK Almene Fund, and Johan and Lise Boserup's Fund....

  12. Lipoprotein(a) levels, isoform size, and risk of type 2 diabetes: A Mendelian randomisation study

    DEFF Research Database (Denmark)

    Kamstrup, Pia Rørbæk; Nordestgaard, Børge G.

    2013-01-01

    study to investigate whether large isoform size, low concentrations in plasma, or both, are causally associated with type 2 diabetes. Methods: We assessed data for adults from the Danish general population enrolled in the Copenhagen City Heart Study and the Copenhagen General Population Study, with and......(a) concentrations alone seem not to be causally associated with type 2 diabetes, but a causal association for large lipoprotein(a) isoform size cannot be excluded. Funding: Danish Heart Foundation, Danish Council for Independent Research-Medical Sciences, IMK Almene Fund, and Johan and Lise Boserup's Fund....

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

    Directory of Open Access Journals (Sweden)

    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

  14. Characterization of PR-10 genes from eight Betula species and detection of Bet v 1 isoforms in birch pollen

    Directory of Open Access Journals (Sweden)

    van't Westende Wendy PC

    2009-03-01

    Full Text Available Abstract Background Bet v 1 is an important cause of hay fever in northern Europe. Bet v 1 isoforms from the European white birch (Betula pendula have been investigated extensively, but the allergenic potency of other birch species is unknown. The presence of Bet v 1 and closely related PR-10 genes in the genome was established by amplification and sequencing of alleles from eight birch species that represent the four subgenera within the genus Betula. Q-TOF LC-MSE was applied to identify which PR-10/Bet v 1 genes are actually expressed in pollen and to determine the relative abundances of individual isoforms in the pollen proteome. Results All examined birch species contained several PR-10 genes. In total, 134 unique sequences were recovered. Sequences were attributed to different genes or pseudogenes that were, in turn, ordered into seven subfamilies. Five subfamilies were common to all birch species. Genes of two subfamilies were expressed in pollen, while each birch species expressed a mixture of isoforms with at least four different isoforms. Isoforms that were similar to isoforms with a high IgE-reactivity (Bet v 1a = PR-10.01A01 were abundant in all species except B. lenta, while the hypoallergenic isoform Bet v 1d (= PR-10.01B01 was only found in B. pendula and its closest relatives. Conclusion Q-TOF LC-MSE allows efficient screening of Bet v 1 isoforms by determining the presence and relative abundance of these isoforms in pollen. B. pendula contains a Bet v 1-mixture in which isoforms with a high and low IgE-reactivity are both abundant. With the possible exception of B. lenta, isoforms identical or very similar to those with a high IgE-reactivity were found in the pollen proteome of all examined birch species. Consequently, these species are also predicted to be allergenic with regard to Bet v 1 related allergies.

  15. Regulation of cell proliferation by nucleocytoplasmic dynamics of postnatal and embryonic exon-II-containing MBP isoforms

    NARCIS (Netherlands)

    Ozgen, Hande; Kahya, Nicoletta; de Jonge, Jenny C.; Smith, Graham S. T.; Harauz, George; Hoekstra, Dick; Baron, Wia

    2014-01-01

    The only known structural protein required for formation of myelin, produced by oligodendrocytes in the central nervous system, is myelin basic protein (MBP). This peripheral membrane protein has different developmentally-regulated isoforms, generated by alternative splicing. The isoforms are target

  16. Lipoprotein(a) levels, apo(a) isoform size, and coronary heart disease risk in the Framingham Offspring Study

    Science.gov (United States)

    The aim of this study was to assess the independent contributions of plasma levels of lipoprotein(a) [Lp(a)], Lp(a) cholesterol, and of apo(a) isoform size to prospective coronary heart disease (CHD) risk. Plasma Lp(a) and Lp(a) cholesterol levels, and apo(a) isoform size were measured at examinati...

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

    Science.gov (United States)

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

    2013-02-28

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

  18. Salivary Gland Thrombostasin Isoforms Differentially Regulate Blood Uptake of Horn Flies Fed on New Zealand White Rabbits

    Science.gov (United States)

    Thrombostasin (TS) is a previously characterized anticlotting protein with multiple isoforms found in the saliva of horn flies. In this report the effects of TS isoforms on blood feeding was assessed with individual flies that carried corresponding ts alleles. Laboratory studies of horn fly blood fe...

  19. Salivary gland thrombostasin isoforms differentilally regulate blood uptake of horn flies fed on New Zealand white rabbits

    Science.gov (United States)

    Thrombostasin (TS) is a previously characterized anticlotting protein with multiple isoforms found in the saliva of horn flies. In this report the effects of TS isoforms on blood feeding was assessed with individual flies that carried corresponding ts alleles. Laboratory studies of horn fly blood fe...

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

    Science.gov (United States)

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

    2016-02-15

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

  1. The characterization of soybean oil body integral oleosin isoforms and the effects of alkaline pH on them.

    Science.gov (United States)

    Cao, Yanyun; Zhao, Luping; Ying, Yusang; Kong, Xiangzhen; Hua, Yufei; Chen, Yeming

    2015-06-15

    Oil body, an organelle in seed cell (naturally pre-emulsified oil), has great potentials to be used in food, cosmetics, pharmaceutical and other applications requiring stable oil-in-water emulsions. Researchers have tried to extract oil body by alkaline buffers, which are beneficial for removing contaminated proteins. But it is not clear whether alkaline buffers could remove oil body integral proteins (mainly oleosins), which could keep oil body integrity and stability. In this study, seven oleosin isoforms were identified for soybean oil body (three isoforms, 24 kDa; three isoforms, 18 kDa; one isoform, 16kDa). Oleosins were not glycoproteins and 24 kDa oleosin isoforms possessed less thiol groups than 18 kDa ones. It was found that alkaline pH not only removed contaminated proteins but also oleosins, and more and more oleosins were removed with increasing alkaline pH.

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

    Science.gov (United States)

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

    2014-10-31

    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. Surprisingly, molecular probes to monitor GalNAc-transferase activity are lacking and there exist no effective global or isoform-specific inhibitors. Here we describe the development of T2- and T3-isoform specific fluorescence sensors that traffic in the secretory pathway. Each sensor yielded little signal when glycosylated but was strongly activated in the absence of its glycosylation. Specificity of each sensor was assessed in HEK cells with either the T2 or T3 enzymes deleted. Although the sensors are based on specific substrates of the T2 and T3 enzymes, elements in or near the enzyme recognition sequence influenced their activity and required modification, which we carried out based on previous in vitro work. Significantly, the modified T2 and T3 sensors were activated only in cells lacking their corresponding isozymes. Thus, we have developed T2- and T3-specific sensors that will be valuable in both the study of GalNAc-transferase regulation and in high-throughput screening for potential therapeutic regulators of specific GalNAc-transferases.

  3. Active zone scaffolds differentially accumulate Unc13 isoforms to tune Ca(2+) channel-vesicle coupling.

    Science.gov (United States)

    Böhme, Mathias A; Beis, Christina; Reddy-Alla, Suneel; Reynolds, Eric; Mampell, Malou M; Grasskamp, Andreas T; Lützkendorf, Janine; Bergeron, Dominique Dufour; Driller, Jan H; Babikir, Husam; Göttfert, Fabian; Robinson, Iain M; O'Kane, Cahir J; Hell, Stefan W; Wahl, Markus C; Stelzl, Ulrich; Loll, Bernhard; Walter, Alexander M; Sigrist, Stephan J

    2016-10-01

    Brain function relies on fast and precisely timed synaptic vesicle (SV) release at active zones (AZs). Efficacy of SV release depends on distance from SV to Ca(2+) channel, but molecular mechanisms controlling this are unknown. Here we found that distances can be defined by targeting two unc-13 (Unc13) isoforms to presynaptic AZ subdomains. Super-resolution and intravital imaging of developing Drosophila melanogaster glutamatergic synapses revealed that the Unc13B isoform was recruited to nascent AZs by the scaffolding proteins Syd-1 and Liprin-α, and Unc13A was positioned by Bruchpilot and Rim-binding protein complexes at maturing AZs. Unc13B localized 120 nm away from Ca(2+) channels, whereas Unc13A localized only 70 nm away and was responsible for docking SVs at this distance. Unc13A(null) mutants suffered from inefficient, delayed and EGTA-supersensitive release. Mathematical modeling suggested that synapses normally operate via two independent release pathways differentially positioned by either isoform. We identified isoform-specific Unc13-AZ scaffold interactions regulating SV-Ca(2+)-channel topology whose developmental tightening optimizes synaptic transmission.

  4. The human-specific invariant chain isoform Iip35 modulates Iip33 trafficking and function.

    Science.gov (United States)

    Sand, Kine Marita Knudsen; Landsverk, Ole J B; Berg-Larsen, Axel; Bakke, Oddmund; Gregers, Tone F

    2014-10-01

    The invariant chain (Ii) is a multifunctional protein, which has an essential role in the assembly and transport of major histocompatibility complex class II (MHC II) molecules. From a single gene, Ii is synthesized as four different isoforms: Iip33, Iip35, Iip41 and Iip43. Iip35 and Iip43 are specific to humans, and are formed due to an upstream alternative translation site, resulting in an N-terminal extension of 16 amino acids. This extension harbors a strong endoplasmic reticulum (ER) retention motif. Consequently, Iip35 or Iip43 expressed alone are retained in the ER, whereas Iip33 and Iip41 rapidly traffic to the endosomal pathway. Endogenously expressed, the four isoforms form mixed heterotrimers in the ER; however, mainly due to the absence of the Iip35/p43 isoforms in mice, little is known about how they influence general Ii function. In this study, we have co-expressed Iip33 and Iip35 in human cells with and without MHC II to gain a better understanding of how Iip35 isoform influences the cellular properties of Iip33. We find that Iip35 significantly affects the properties of Iip33. In the presence of Iip35, the transport of Iip33 out of the ER is delayed, its half-life is dramatically prolonged and its ability to induce enlarged endosomes and delayed endosomal maturation is abrogated.

  5. Tyrosinase activity and isoform composition in separate tissues during development of Agaricus bisporus fruit bodies

    NARCIS (Netherlands)

    Leeuwen, van J.; Wichers, H.J.

    1999-01-01

    During growth of Agaricus bisporus fruit bodies the amount of active tyrosinase increased. The amount of active tyrosinase can be related to the degree of browning, as opposed to the fully activated tyrosinase level. Isoelectric focusing revealed that active and latent tyrosinase isoforms having dif

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  8. Alternative NF-κB Isoforms in the Drosophila Neuromuscular Junction and Brain.

    Directory of Open Access Journals (Sweden)

    Bo Zhou

    Full Text Available The Drosophila NF-κB protein Dorsal is expressed at the larval neuromuscular junction, where its expression appears unrelated to known Dorsal functions in embryonic patterning and innate immunity. Using confocal microscopy with domain-specific antisera, we demonstrate that larval muscle expresses only the B isoform of Dorsal, which arises by intron retention. We find that Dorsal B interacts with and stabilizes Cactus at the neuromuscular junction, but exhibits Cactus independent localization and an absence of detectable nuclear translocation. We further find that the Dorsal-related immune factor Dif encodes a B isoform, reflecting a conservation of B domains across a range of insect NF-κB proteins. Carrying out mutagenesis of the Dif locus via a site-specific recombineering approach, we demonstrate that Dif B is the major, if not sole, Dif isoform in the mushroom bodies of the larval brain. The Dorsal and Dif B isoforms thus share a specific association with nervous system tissues as well as an alternative protein structure.

  9. Recombinant heptameric coatomer complexes: novel tools to study isoform-specific functions.

    NARCIS (Netherlands)

    Sahlmuller, M.C.; Strating, J.R.P.M.; Beck, R.; Eckert, P.; Popoff, V.; Haag, M.; Hellwig, A.; Berger, I.; Brugger, B.; Wieland, F.T.

    2011-01-01

    COPI (coat protein I)-coated vesicles are implicated in various transport steps within the early secretory pathway. The major structural component of the COPI coat is the heptameric complex coatomer (CM). Recently, four isoforms of CM were discovered that may help explain various transport steps in

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

    Science.gov (United States)

    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.

  11. Synthesis of Benzofuran Analogue of Go6976, an Isoform Selective Protein Kinase C Inhibitor

    Institute of Scientific and Technical Information of China (English)

    MA, Da-Wei; ZHANG, Xin-Rong; WU, Shi-Hui; TAO, Feng-Gang

    2001-01-01

    Based on the structure of Go6976, a known isoform-selective protein kinase C inhibitor, a benzofuran analogue (1) was designed. This analogue was synthesized by coupling of benzofuran 3-acetic acid and 8-oxo-tryptamine and subsequent intramolecular Dieckmann condensation, alkylation, oxidative photocyclization and cyanation reaction of mesylate.

  12. Functions of Glutamine Synthetase Isoforms in the Nitrogen Metabolism of Plants

    DEFF Research Database (Denmark)

    Guan, Miao

    ;2 which encode different isoforms of the key N-assimilatory enzyme cytosolic glutamine synthetase (GS1). In the single knockout mutant gln1;2 and in the double knockout mutant gln1;1:gln1;2, seed germination and seedling establishment were distinctly impaired. The negative effect of Gln1;2 deficiency...

  13. Translational control of SCL-isoform expression in hematopoietic lineage choice

    NARCIS (Netherlands)

    Calkhoven, Cornelis F; Muller, Christine; Martin, Richard; Krosl, Goradz; Pietsch, Hubertus; Hoang, Trang; Leutz, Achim

    2003-01-01

    We investigated the translational regulation of SCL protein expression and its role in hematopoietic lineage choice. We show that the expression of different SCL protein isoforms is regulated by signal transduction pathways that modulate translation initiation factor (eIF) function. A conserved smal

  14. Improved mass spectrometry assay for plasma hepcidin: detection and characterization of a novel hepcidin isoform

    NARCIS (Netherlands)

    Laarakkers, C.M.; Wiegerinck, E.T.G.; Klaver, S.; Kolodziejczyk, M.; Gille, H.; Hohlbaum, A.M.; Tjalsma, H.; Swinkels, D.W.

    2013-01-01

    Mass spectrometry (MS)-based assays for the quantification of the iron regulatory hormone hepcidin are pivotal to discriminate between the bioactive 25-amino acid form that can effectively block the sole iron transporter ferroportin and other naturally occurring smaller isoforms without a known role

  15. Cloning, Sequencing, and the Expression of the Elusive Sarcomeric TPM4α Isoform in Humans

    Science.gov (United States)

    Abbott, Lynn; Alshiekh-Nasany, Ruham; Mitschow, Charles

    2016-01-01

    In mammals, tropomyosin is encoded by four known TPM genes (TPM1, TPM2, TPM3, and TPM4) each of which can generate a number of TPM isoforms via alternative splicing and/or using alternate promoters. In humans, the sarcomeric isoform(s) of each of the TPM genes, except for the TPM4, have been known for a long time. Recently, on the basis of computational analyses of the human genome sequence, the predicted sequence of TPM4α has been posted in GenBank. We designed primer-pairs for RT-PCR and showed the expression of the transcripts of TPM4α and a novel isoform TPM4δ in human heart and skeletal muscle. qRT-PCR shows that the relative expression of TPM4α and TPM4δ is higher in human cardiac muscle. Western blot analyses using CH1 monoclonal antibodies show the absence of the expression of TPM4δ protein (~28 kDa) in human heart muscle. 2D western blot analyses with the same antibody show the expression of at least nine distinct tropomyosin molecules with a mass ~32 kD and above in adult heart. By Mass spectrometry, we determined the amino acid sequences of the extracted proteins from these spots. Spot “G” reveals the putative expression of TPM4α along with TPM1α protein in human adult heart. PMID:27703814

  16. Cloning, Sequencing, and the Expression of the Elusive Sarcomeric TPM4α Isoform in Humans

    Directory of Open Access Journals (Sweden)

    Dipak K. Dube

    2016-01-01

    Full Text Available In mammals, tropomyosin is encoded by four known TPM genes (TPM1, TPM2, TPM3, and TPM4 each of which can generate a number of TPM isoforms via alternative splicing and/or using alternate promoters. In humans, the sarcomeric isoform(s of each of the TPM genes, except for the TPM4, have been known for a long time. Recently, on the basis of computational analyses of the human genome sequence, the predicted sequence of TPM4α has been posted in GenBank. We designed primer-pairs for RT-PCR and showed the expression of the transcripts of TPM4α and a novel isoform TPM4δ in human heart and skeletal muscle. qRT-PCR shows that the relative expression of TPM4α and TPM4δ is higher in human cardiac muscle. Western blot analyses using CH1 monoclonal antibodies show the absence of the expression of TPM4δ protein (~28 kDa in human heart muscle. 2D western blot analyses with the same antibody show the expression of at least nine distinct tropomyosin molecules with a mass ~32 kD and above in adult heart. By Mass spectrometry, we determined the amino acid sequences of the extracted proteins from these spots. Spot “G” reveals the putative expression of TPM4α along with TPM1α protein in human adult heart.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. Titin isoforms and kinematics of fast swimming carp larvae (Cyprinus carpio L.)

    NARCIS (Netherlands)

    Spierts, I.L.Y.

    2001-01-01

    Titin, a striated-muscle specific protein spanning the distance between Z- and M-lines of sarcomeres, is held responsible for developing passive tension and for maintaining the central position of thick filaments in contracting sarcomeres. Different muscles express titin isoforms of different molecu

  19. Squamous cell carcinoma antigen isoforms in serum from cervical cancer patients

    NARCIS (Netherlands)

    Roijer, E; de Bruijn, HWA; Dahlen, U; ten Hoor, K; Lundin, M; Nilsson, K; Soderstrom, K; Nilsson, O

    2006-01-01

    Squamous cell carcinoma antigen (SCCA) is a serological marker of squamous cell carcinomas (SCC). To study whether any of the SCCA isoforms would provide additional and more specific/sensitive clinical information than total SCCA, immunoassays specific for the different forms of SCCA (free SCCA2, to

  20. Role of PRMTs in cancer: Could minor isoforms be leaving a mark?

    Institute of Scientific and Technical Information of China (English)

    R; Mitchell; Baldwin; Alan; Morettin; Jocelyn; C?té

    2014-01-01

    Protein arginine methyltransferases(PRMTs) catalyze the methylation of a variety of protein substrates, many of which have been linked to the development, progression and aggressiveness of different types of cancer. Moreover, aberrant expression of PRMTs has been observed in several cancer types. While the link between PRMTs and cancer is a relatively new area of interest, the functional implications documented thus far warrant further investigations into its therapeutic potential. However, the expression of these enzymes and the regulation of their activity in cancer are still significantly understudied. Currently there are nine main members of the PRMT family. Further, the existence of alternatively spliced isoforms for several of these family members provides an additional layer of complexity. Specifically, PRMT1, PRMT2, CARM1 and PRMT7 have been shown to have alternative isoforms and others may be currently unrealized. Our knowledge with respect to the relative expression and the specific functions of these isoforms is largely lacking and needs attention. Here we present a review of the current knowledge of theknown alternative PRMT isoforms and provide a rationale for how they may impact on cancer and represent potentially useful targets for the development of novel therapeutic strategies.

  1. Effect of renal replacement therapy on retinol-binding protein 4 isoforms

    DEFF Research Database (Denmark)

    Frey, Simone K; Henze, Andrea; Nagl, Britta;

    2009-01-01

    Retinol-binding protein 4 (RBP4) levels are elevated in the serum of patients with kidney dysfunction. We recently showed that RBP4 isoforms including apo-RBP4 (RBP4 not bound to retinol) and RBP4 truncated at the C-terminus (RBP4-L, RBP4-LL) are increased in the serum of patients with kidney dis...

  2. Diagnostic Accuracy of Cerebrospinal Fluid Amyloid-beta Isoforms for Early and Differential Dementia Diagnosis

    NARCIS (Netherlands)

    Struyfs, Hanne; Van Broeck, Bianca; Timmers, Maarten; Fransen, Erik; Sleegers, Kristel; Van Broeckhoven, Christine; De Deyn, Peter P.; Streffer, Johannes R.; Mercken, Marc; Engelborghs, Sebastiaan

    2015-01-01

    Background: Overlapping cerebrospinal fluid biomarkers (CSF) levels between Alzheimer's disease (AD) and non-AD patients decrease differential diagnostic accuracy of the AD core CSF biomarkers. Amyloid-beta (A beta) isoforms might improve the AD versus non-AD differential diagnosis. Objective: To de

  3. Differential expression of polycytosine-binding protein isoforms in adrenal gland, locus coeruleus and midbrain.

    Science.gov (United States)

    Boschi, N M; Takeuchi, K; Sterling, C; Tank, A W

    2015-02-12

    Polycytosine-binding proteins (PCBPs) are RNA-binding proteins that participate in post-transcriptional control pathways. Among the diverse functions of these proteins is the interaction with a 27 nucleotide pyrimidine-rich domain within the 3'UTR of tyrosine hydroxylase (TH) mRNA. Mutations to this domain result in decreased stability of TH mRNA and loss of cAMP-mediated activation of TH mRNA translation. PCBPs are hypothesized to play key roles in these regulatory mechanisms. In order to further test this hypothesis, we examined the tissue distribution of PCBPs in catecholaminergic cells. Initial studies demonstrated that proteins from catecholaminergic tissues bind to TH mRNA 3'UTR sequences and these proteins have an apparent Mr of ∼ 44 kDa, which is close to the molecular sizes for PCBPs. Fluorescent immunohistochemistry and confocal microscopy was used to analyze the distribution of PCBP isoforms in TH-positive cells of the rat midbrain, locus coeruleus, and adrenal gland. Our results suggest that: (1) PCBP2 is the predominant isoform in TH-positive cells of the rat midbrain; (2) PCBP3 is the predominant isoform in TH-positive cells of the locus coeruleus; and (3) PCBP1 is the predominant isoform in the adrenal medulla. The localization of PCBP proteins to TH-positive cells in these catecholaminergic tissues is consistent with the hypothesis that PCBPs play a role in the regulation of TH expression.

  4. Heparanase isoform expression and extracellular matrix remodeling in intervertebral disc degenerative disease

    Directory of Open Access Journals (Sweden)

    Luciano Miller Reis Rodrigues

    2011-01-01

    Full Text Available OBJECTIVE: To determine the molecules involved in extracellular matrix remodeling and to identify and quantify heparanase isoforms present in herniated and degenerative discs. INTRODUCTION: Heparanase is an endo-beta-glucuronidase that specifically acts upon the heparan sulfate chains of proteoglycans. However, heparanase expression in degenerative intervertebral discs has not yet been evaluated. Notably, previous studies demonstrated a correlation between changes in the heparan sulfate proteoglycan pattern and the degenerative process associated with intervertebral discs. METHODS: Twenty-nine samples of intervertebral degenerative discs, 23 samples of herniated discs and 12 samples of non-degenerative discs were analyzed. The expression of both heparanase isoforms (heparanase-1 and heparanase-2 was evaluated using immunohistochemistry and real-time RT-PCR analysis. RESULTS: Heparanase-1 and heparanase-2 expression levels were significantly higher in the herniated and degenerative discs in comparison to the control tissues, suggesting a possible role of these proteins in the intervertebral degenerative process. CONCLUSION: The overexpression of heparanase isoforms in the degenerative intervertebral discs and the herniated discs suggests a potential role of both proteins in the mediation of inflammatory processes and in extracellular matrix remodeling. The heparanase-2 isoform may be involved in normal metabolic processes, as evidenced by its higher expression in the control intervertebral discs relative to the expression of heparanase-1.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. The endogenous anti-angiogenic VEGF isoform, VEGF165b inhibits human tumour growth in mice.

    NARCIS (Netherlands)

    Rennel, E.; Waine, E.; Guan, H.; Schuler, Y.; Leenders, W.P.J.; Woolard, J.; Sugiono, M.; Gillatt, D.; Kleinerman, E.; Bates, D.; Harper, S.

    2008-01-01

    Vascular endothelial growth factor-A is widely regarded as the principal stimulator of angiogenesis required for tumour growth. VEGF is generated as multiple isoforms of two families, the pro-angiogenic family generated by proximal splice site selection in the terminal exon, termed VEGFxxx, and the

  7. Plectin isoform 1b mediates mitochondrion-intermediate filament network linkage and controls organelle shape.

    Science.gov (United States)

    Winter, Lilli; Abrahamsberg, Christina; Wiche, Gerhard

    2008-06-16

    Plectin is a versatile intermediate filament (IF)-bound cytolinker protein with a variety of differentially spliced isoforms accounting for its multiple functions. One particular isoform, plectin 1b (P1b), remains associated with mitochondria after biochemical fractionation of fibroblasts and cells expressing exogenous P1b. Here, we determined that P1b is inserted into the outer mitochondrial membrane with the exon 1b-encoded N-terminal sequence serving as a mitochondrial targeting and anchoring signal. To study P1b-related mitochondrial functions, we generated mice that selectively lack this isoform but express all others. In primary fibroblasts and myoblasts derived from these mice, we observe a substantial elongation of mitochondrial networks, whereas other mitochondrial properties remain largely unaffected. Normal morphology of mitochondria could be restored by isoform-specific overexpression of P1b in P1b-deficient as well as plectin-null cells. We propose a model where P1b both forms a mitochondrial signaling platform and affects organelle shape and network formation by tethering mitochondria to IFs.

  8. Fatal Hepatitis Mediated by TNFα Requires Caspase-8 and Involves the BH3-only Proteins Bid and Bim

    OpenAIRE

    Kaufmann, Thomas; Jost, Philipp J; Pellegrini, Marc; Puthalakath, Hamsa; Gugasyan, Raffi; Gerondakis, Steve; Cretney, Erika; Smyth, Mark J.; Silke, John; Hakem, Razq; Bouillet, Philippe; Mak, Tak W; Dixit, Vishva M.; Strasser, Andreas

    2009-01-01

    Apoptotic death of hepatocytes, a feature and contributing factor of many chronic and acute liver diseases, can be a consequence of over-activation of the immune system. Injection with lipopolysaccharide (LPS) plus the transcriptional inhibitor D(+)-galactosamine (GalN) or mitogenic T cell activation cause fatal hepatocyte apoptosis in mice. In both settings hepatocyte killing is mediated by TNFα/TNF-R signaling but the effector mechanisms remain unclear. Our analysis of gene-targeted mice sh...

  9. Selective expression of myosin IC Isoform A in mouse and human cell lines and mouse prostate cancer tissues.

    Directory of Open Access Journals (Sweden)

    Ivanna Ihnatovych

    Full Text Available Myosin IC is a single headed member of the myosin superfamily. We recently identified a novel isoform and showed that the MYOIC gene in mammalian cells encodes three isoforms (isoforms A, B, and C. Furthermore, we demonstrated that myosin IC isoform A but not isoform B exhibits a tissue specific expression pattern. In this study, we extended our analysis of myosin IC isoform expression patterns by analyzing the protein and mRNA expression in various mammalian cell lines and in various prostate specimens and tumor tissues from the transgenic mouse prostate (TRAMP model by immunoblotting, qRT-PCR, and by indirect immunohistochemical staining of paraffin embedded prostate specimen. Analysis of a panel of mammalian cell lines showed an increased mRNA and protein expression of specifically myosin IC isoform A in a panel of human and mouse prostate cancer cell lines but not in non-cancer prostate or other (non-prostate- cancer cell lines. Furthermore, we demonstrate that myosin IC isoform A expression is significantly increased in TRAMP mouse prostate samples with prostatic intraepithelial neoplasia (PIN lesions and in distant site metastases in lung and liver when compared to matched normal tissues. Our observations demonstrate specific changes in the expression of myosin IC isoform A that are concurrent with the occurrence of prostate cancer in the TRAMP mouse prostate cancer model that closely mimics clinical prostate cancer. These data suggest that elevated levels of myosin IC isoform A may be a potential marker for the detection of prostate cancer.

  10. Conformational difference in human IgG2 disulfide isoforms revealed by hydrogen/deuterium exchange mass spectrometry.

    Science.gov (United States)

    Zhang, Aming; Fang, Jing; Chou, Robert Y-T; Bondarenko, Pavel V; Zhang, Zhongqi

    2015-03-17

    Both recombinant and natural human IgG2 antibodies have several different disulfide bond isoforms, which possess different global structures, thermal stabilities, and biological activities. A detailed mapping of the structural difference among IgG2 disulfide isoforms, however, has not been established. In this work, we employed hydrogen/deuterium exchange mass spectrometry to study the conformation of three major IgG2 disulfide isoforms known as IgG2-B, IgG2-A1, and IgG2-A2 in two recombinant human IgG2 monoclonal antibodies. By comparing the protection factors between amino acid residues in isoforms B and A1 (the classical form), we successfully identified several local regions in which the IgG2-B isoform showed more solvent protection than the IgG2-A1 isoform. On the basis of three-dimensional structural models of IgG2, these identified regions were located on the Fab domains, close to the hinge, centered on the side where the two Fab arms faced each other in spatial proximity. We speculated that in the more solvent-protected B isoform, the two Fab arms were brought into contact by the nonclassical disulfide bonds, resulting in a more compact global structure. Loss of Fab domain flexibility in IgG2-B could limit its ability to access cell-surface epitopes, leading to reduced antigen binding potency. The A2 isoform was previously found to have disulfide linkages similar to those of the classical A1 isoform, but with different biophysical behaviors. Our data indicated that, compared to IgG2-A1, IgG2-A2 had less solvent protection in some heavy-chain Fab regions close the hinge, suggesting that the A2 isoform had more flexible Fab domains. PMID:25730439

  11. mRNA expression of diacylglycerol kinase isoforms in insulin-sensitive tissues: effects of obesity and insulin resistance.

    Science.gov (United States)

    Mannerås-Holm, Louise; Kirchner, Henriette; Björnholm, Marie; Chibalin, Alexander V; Zierath, Juleen R

    2015-04-01

    Diacylglycerol kinase (DGK) isoforms regulate signal transduction and lipid metabolism. DGKδ deficiency leads to hyperglycemia, peripheral insulin resistance, and metabolic inflexibility. Thus, dysregulation of other DGK isoforms may play a role in metabolic dysfunction. We investigated DGK isoform mRNA expression in extensor digitorum longus (EDL) and soleus muscle, liver as well as subcutaneous and epididymal adipose tissue in C57BL/6J mice and obese and insulin-resistant ob/ob mice. All DGK isoforms, except for DGKκ, were detectable, although with varying mRNA expression. Liver DGK expression was generally lowest, with several isoforms undetectable. In soleus muscle, subcutaneous and epididymal adipose tissue, DGKδ was the most abundant isoform. In EDL muscle, DGKα and DGKζ were the most abundant isoforms. In liver, DGKζ was the most abundant isoform. Comparing obese insulin-resistant ob/ob mice to lean C57BL/6J mice, DGKβ, DGKι, and DGKθ were increased and DGKε expression was decreased in EDL muscle, while DGKβ, DGKη and DGKθ were decreased and DGKδ and DGKι were increased in soleus muscle. In liver, DGKδ and DGKζ expression was increased in ob/ob mice. DGKη was increased in subcutaneous fat, while DGKζ was increased and DGKβ, DGKδ, DGKη and DGKε were decreased in epididymal fat from ob/ob mice. In both adipose tissue depots, DGKα and DGKγ were decreased and DGKι was increased in ob/ob mice. In conclusion, DGK mRNA expression is altered in an isoform- and tissue-dependent manner in obese insulin-resistant ob/ob mice. DGK isoforms likely have divergent functional roles in distinct tissues, which may contribute to metabolic dysfunction. PMID:25847921

  12. Two Distinct Isoforms of Matrix Metalloproteinase-2 Are Associated with Human Delayed Kidney Graft Function.

    Directory of Open Access Journals (Sweden)

    Shaynah Wanga

    Full Text Available Delayed graft function (DGF is a frequent complication of renal transplantation, particularly in the setting of transplantation of kidneys derived from deceased donors and expanded-criteria donors. DGF results from tubular epithelial cell injury and has immediate and long term consequences. These include requirement for post-transplantation dialysis, increased incidence of acute rejection, and poorer long-term outcomes. DGF represents one of the clearest clinical examples of renal acute ischemia/reperfusion injury. Experimental studies have demonstrated that ischemia/reperfusion injury induces the synthesis of the full length secreted isoform of matrix metalloproteinase-2 (FL-MMP-2, as well as an intracellular N-terminal truncated MMP-2 isoform (NTT-MMP-2 that initiates an innate immune response. We hypothesized that the two MMP-2 isoforms mediate tubular epithelial cell injury in DGF. Archival renal biopsy sections from 10 protocol biopsy controls and 41 cases with a clinical diagnosis of DGF were analyzed for the extent of tubular injury, expression of the FL-MMP-2 and NTT-MMP-2 isoforms by immunohistochemistry (IHC, in situ hybridization, and qPCR to determine isoform abundance. Differences in transcript abundance were related to tubular injury score. Markers of MMP-2-mediated injury included TUNEL staining and assessment of peritubular capillary density. There was a clear relationship between tubular epithelial cell expression of both FL-MMP-2 and NTT-MMP-2 IHC with the extent of tubular injury. The MMP-2 isoforms were detected in the same tubular segments and were present at sites of tubular injury. qPCR demonstrated highly significant increases in both the FL-MMP-2 and NTT-MMP-2 transcripts. Statistical analysis revealed highly significant associations between FL-MMP-2 and NTT-MMP-2 transcript abundance and the extent of tubular injury, with NTT-MMP-2 having the strongest association. We conclude that two distinct MMP-2 isoforms are

  13. Kalrn promoter usage and isoform expression respond to chronic cocaine exposure

    Directory of Open Access Journals (Sweden)

    Ma Xin-Ming

    2011-02-01

    Full Text Available Abstract Background The long-term effects of cocaine on behavior are accompanied by structural changes in excitatory glutamatergic synapses onto the medium spiny neurons of the striatum. The Kalrn gene encodes several functionally distinct isoforms; these multidomain guanine nucleotide exchange factors (GEFs contain additional domains known to interact with phosphatidylinositides as well as with a number of different proteins. Through their activation of Rho proteins and their interactions with other proteins, the different Kalirin isoforms affect cytoskeletal organization. Chronic exposure of adult male rodents to cocaine increases levels of Kalirin 7 in the striatum. When exposed chronically to cocaine, mice lacking Kalirin 7, the major adult isoform, fail to show an increase in dendritic spine density in the nucleus accumbens, show diminished place preference for cocaine, and exhibit increased locomotor activity in response to cocaine. Results The use of alternate promoters and 3'-terminal exons of the mouse Kalrn gene were investigated using real-time quantitative polymerase chain reaction. While the two most distal full-length Kalrn promoters are used equally in the prefrontal cortex, the more proximal of these promoters accounts for most of the transcripts expressed in the nucleus accumbens. The 3'-terminal exon unique to the Kalirin 7 isoform accounts for a greater percentage of the Kalrn transcripts in prefrontal cortex than in nucleus accumbens. Western blot analyses confirmed these differences. Chronic cocaine treatment increases usage of the promoter encoding the Δ-Kalirin isoforms but does not alter full-length Kalirin promoter usage. Usage of the 3'-terminal exon unique to Kalirin 7 increases following chronic cocaine exposure. Conclusions Kalrn promoter and 3'-terminal exon utilization are region-specific. In the nucleus accumbens, cocaine-mediated alterations in promoter usage and 3'-terminal exon usage favor expression of

  14. Arabidopsis Profilin Isoforms, PRF1 and PRF2Show Distinctive Binding Activities and Subcellular Distributions

    Institute of Scientific and Technical Information of China (English)

    Feng Wang; Yanping Jing; Zhen Wang; Tonglin Mao; Jozef (S)amaj; Ming Yuan; Haiyun Ren

    2009-01-01

    Profilin is an actin-binding protein that shows complex effects on the dynamics of the actin cytoskeleton. There are five profilin isoforms in Arabidopsis thaliana L. However, it is still an open question whether these isoforms are functionally different. In the present study, two profilin isoforms from Arabidopsis, PRF1 and PRF2 were fused with green fluorescent protein (GFP) tag and expressed in Escherichia coli and A. thaliana in order to compare their biochemical properties in vitro and their cellular distributions in vivo. Biochemical analysis revealed that fusion proteins of GFP-PRF1 and GFP-PRF2 can bind to poly-L-proline and G-actin showing remarkable differences. GFP-PRF1 has much higher affinities for both poly-L-proline and G-actin compared with GFP-PRF2. Observations of living cells in stable transgsnic A. thaliana lines revealed that 35S::GFP-PRF1 formed a filamentous network, while 35S::GFP-PRF2 formed polygonal meshes. Results from the treatment with latrunculin A and a subsequent recovery experiment indicated that filamentous alignment of GFP-PRF1 was likely associated with actin filaments. However, GFP-PRF2 localized to polygonal meshes resembling the endoplasmic reticulum. Our results provide evidence that Arabidopsis profllin isoforms PRF1 and PRF2 have different biochemical affinities for poly-L-proline and G-actin, and show distinctive Iocalizations in living cells. These data suggest that PRF1 and PRF2 are functionally different isoforms.

  15. Survivin isoforms and clinicopathological characteristics in colorectal adenocarcinomas using real-time qPCR

    Science.gov (United States)

    Pavlidou, Anastasia; Dalamaga, Maria; Kroupis, Christos; Konstantoudakis, George; Belimezi, Maria; Athanasas, George; Dimas, Kleanthi

    2011-01-01

    AIM: To investigate three isoforms of survivin in colorectal adenocarcinomas. METHODS: We used the LightCycler Technology (Roche), along with a common forward primer and reverse primers specific for the splice variants and two common hybridization probes labeled with fluorescein and LightCycler-Red fluorophore (LC-Red 640). Real time quantitative polymerase chain reaction (PCR) was performed on cDNAs from 52 tumor specimens from colorectal cancer patients and 10 unrelated normal colorectal tissues. In the patients group, carcinoembryonic antigen (CEA) and CA19-9 tumor markers were also measured immunochemically. RESULTS: Wild type survivin mRNA isoform was expressed in 48% of the 52 tumor samples, survivin-2b in 38% and survivin-ΔΕx3 in 29%, while no expression was found in normal tissues. The mRNA expression of wild type survivin presented a significant correlation with the expression of the ratio of survivin-2b, survivin-ΔΕx3, survivin-2b/wild type survivin and survivin-ΔΕx3/wild type survivin (P < 0.001). The mRNA expression of wild-survivin and survivin-ΔΕx3 was related with tumor size and invasion (P = 0.006 and P < 0.005, respectively). A significant difference was found between survivin-2b and morphologic cancer type. Also, the ratio of survivin-ΔEx3/wild-survivin was significantly associated with prognosis. No association was observed between the three isoforms and grade, metastasis, Dukes stage and gender. The three isoforms were not correlated with CEA and CA19-9. CONCLUSION: Survivin isoforms may play a role in cell apoptosis and their quantification could provide information about clinical management of patients suffering from colorectal cancer. PMID:21472129

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  17. Knockout mutants as a tool to identify the subunit composition of Arabidopsis glutamine synthetase isoforms.

    Science.gov (United States)

    Dragićević, Milan; Todorović, Slađana; Bogdanović, Milica; Filipović, Biljana; Mišić, Danijela; Simonović, Ana

    2014-06-01

    Glutamine synthetase (GS) is a key enzyme in nitrogen assimilation, which catalyzes the formation of glutamine from ammonia and glutamate. Plant GS isoforms are multimeric enzymes, recently shown to be decamers. The Arabidopsis genome encodes five cytosolic (GS1) proteins labeled as GLN1;1 through GLN1;5 and one chloroplastic (GS2) isoform, GLN2;0. However, as many as 11 GS activity bands were resolved from different Arabidopsis tissues by Native PAGE and activity staining. Western analysis showed that all 11 isoforms are composed exclusively of 40 kDa GS1 subunits. Of five GS1 genes, only GLN1;1, GLN1;2 and GLN1;3 transcripts accumulated to significant levels in vegetative tissues, indicating that only subunits encoded by these three genes produce the 11-band zymogram. Even though the GS2 gene also had significant expression, the corresponding activity was not detected, probably due to inactivation. To resolve the subunit composition of 11 active GS1 isoforms, homozygous knockout mutants deficient in the expression of different GS1 genes were selected from the progeny of T-DNA insertional SALK and SAIL lines. Comparison of GS isoenzyme patterns of the selected GS1 knockout mutants indicated that all of the detected isoforms consist of varying proportions of GLN1;1, GLN1;2 and GLN1;3 subunits, and that GLN1;1 and GLN1;3, as well as GLN1;2 and GLN1;3 and possibly GLN1;1 and GLN1;2 proteins combine in all proportions to form active homo- and heterodecamers.

  18. Muscle Specific Fragile X Related Protein 1 Isoforms are Sequestered in the Nucleus of Undifferentiated Myoblast

    Directory of Open Access Journals (Sweden)

    Khandjian Edouard W

    2000-12-01

    Full Text Available Abstract Background The family of Fragile X Mental Retardation Proteins is composed of three members: Fragile Mental Retardation 1, Fragile X Related 1 and X Related 2 proteins. These proteins are associated with mRNPs within translating ribosomes and have the capacity to shuttle between the nucleus and the cytoplasm. Great attention has been given to FMRP due to its implication in human hereditary mental retardation while FXR1P and FXR2P have only recently been studied. Results Using antibodies directed against several epitopes of FXR1P, we have detected protein isoforms generated by small peptides pocket inserts. Four isoforms of MW 70, 74, 78, 80 kDa are widely distributed in mouse organs, while in striated muscles these isoforms are replaced by proteins of 82 and 84 kDa containing an extra pocket of 27 aa. Expression of these muscle isoforms is an early event during in vitro differentiation of myoblasts into myotubes and correlates with the activation of muscle-specific genes. However, while FXR1P82,84 are associated with cytoplasmic mRNPs in myotubes, they are sequestered in the nuclei of undifferentiated myoblasts. These observations suggest that, in addition to a cytoplasmic function yet to be defined, FXR1P82,84 may play a nuclear role in pre-mRNA metabolism. Conclusions The pattern of subcellular partitioning of FXR1P isoforms during myogenesis is unique among the family of the FXR proteins. The model system described here should be considered as a powerful tool for ongoing attempts to unravel structure-function relationships of the different FMR family members since the potential role(s of FXR1P as a compensatory factor in Fragile X syndrome is still elusive.

  19. Survivin isoforms and clinicopathological characteristics in colorectal adenocarcinomas using real-time qPCR

    Institute of Scientific and Technical Information of China (English)

    Anastasia Pavlidou; Maria Dalamaga; Christos Kroupis; George Konstantoudakis; Maria Belimezi; George Athanasas; Kleanthi Dimas

    2011-01-01

    AIM: To investigate three isoforms of survivin in colorectal adenocarcinomas. METHODS: We used the LightCycler Technology (Roche), along with a common forward primer and reverse primers specific for the splice variants and two common hybridization probes labeled with fluorescein and LightCycler- Red fluorophore (LC-Red 640). Real time quantitative polymerase chain reaction (PCR) was performed on cDNAs from 52 tumor specimens from colorectal cancer patients and 10 unrelated normal colorectal tissues. In the patients group, carcinoembryonic antigen (CEA) and CA19-9 tumor markers were also measured immunochemically. RESULTS: Wild type survivin mRNA isoform was expressed in 48% of the 52 tumor samples, survivin-2b in 38% and survivin-ΔΕx3 in 29%, while no expression was found in normal tissues. The mRNA expression of wild type survivin presented a significant correlation with the expression of the ratio of survivin-2b, survivin-ΔΕx3, survivin-2b/wild type survivin and survivin-ΔΕx3/wild type survivin (P < 0.001). The mRNA expression of wildsurvivin and survivin-ΔΕx3 was related with tumor size and invasion (P = 0.006 and P < 0.005, respectively). A significant difference was found between survivin-2b and morphologic cancer type. Also, the ratio of survivin-ΔEx3/ wild-survivin was significantly associated with prognosis. No association was observed between the three isoforms and grade, metastasis, Dukes stage and gender. The three isoforms were not correlated with CEA and CA19-9. CONCLUSION: Survivin isoforms may play a role in cell apoptosis and their quantification could provide information about clinical management of patients suffering from colorectal cancer.

  20. Changes in type II procollagen isoform expression during chondrogenesis by disruption of an alternative 5’ splice site within Col2a1 exon 2

    OpenAIRE

    Hering, Thomas M.; Wirthlin, Louisa; Ravindran, Soumya; McAlinden, Audrey

    2014-01-01

    This study describes a new mechanism controlling the production of alternatively-spliced isoforms of type II procollagen (Col2a1) in vivo. During chondrogenesis, precursor chondrocytes predominantly produce isoforms containing alternatively-spliced exon 2 (type IIA and IID) while Col2a1 mRNA devoid of exon 2 (type IIB) is the major isoform produced by differentiated chondrocytes. We previously identified an additional Col2a1 isoform containing a truncated exon 2 and premature termination codo...

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

    International Nuclear Information System (INIS)

    Crystallization of important glycoenzymes involved in IgE-mediated latex allergy. Latex from Hevea brasiliensis contains several allergenic proteins that are involved in type I allergy. One of them is Hev b 2, which is a β-1,3-glucanase enzyme that exists in different isoforms with variable glycosylation content. Two glucanase isoforms were isolated from trees of the GV-42 clone by gel filtration, affinity and ion-exchange chromatography. Isoform I had a carbohydrate content of about 20%, with N-linked N-acetyl-glucosamine, N-acetyl-galactosamine, fucose and galactose residues as the main sugars, while isoform II showed 6% carbohydrate content constisting of N-acetyl-glucosamine, fucose, mannose and xylose. Both isoforms were crystallized by the hanging-drop vapour-diffusion method. Isoform I crystals were grown using 0.2 M trisodium citrate dihydrate, 0.1 M Na HEPES pH 7.5 and 20%(v/v) 2-propanol, but these crystals were not appropriate for data collection. Isoform II crystals were obtained under two conditions and X-ray diffraction data were collected from both. In the first condition (0.2 M trisodium citrate, 0.1 M sodium cacodylate pH 6.5, 30% 2-propanol), crystals belonging to the tetragonal space group P41 with unit-cell parameters a = b = 150.17, c = 77.41 Å were obtained. In the second condition [0.2 M ammonium acetate, 0.1 M trisodium citrate dihydrate pH 5.6, 30%(w/v) polyethylene glycol 4000] the isoform II crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 85.08, b = 89.67, c = 101.80 Å, β = 113.6°. Preliminary analysis suggests that there are four molecules of isoform II in both asymmetric units

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

    Energy Technology Data Exchange (ETDEWEB)

    Fuentes-Silva, D. [Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Cuidad Universitaria, Coyoacán, México, DF 04510 (Mexico); Mendoza-Hernández, G. [Facultad de Medicina, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Cuidad Universitaria, Coyoacán, México, DF 04510 (Mexico); Stojanoff, V. [Brookhaven National Laboratory, National Synchrotron Light Source, Upton, NY (United States); Palomares, L. A. [Instituto de Biotecnología, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Cuidad Universitaria, Coyoacán, México, DF 04510 (Mexico); Zenteno, E. [Facultad de Medicina, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Cuidad Universitaria, Coyoacán, México, DF 04510 (Mexico); Torres-Larios, A. [Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Cuidad Universitaria, Coyoacán, México, DF 04510 (Mexico); Rodríguez-Romero, A., E-mail: adela@servidor.unam.mx [Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Cuidad Universitaria, Coyoacán, México, DF 04510 (Mexico)

    2007-09-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Fuentes-Silva,D.; Mendoza-Hernandez, G.; Stojanoff, V.; Palomares, L.; Zenteno, E.; Torres-Larios, A.; Rodriguez-Romero, A.

    2007-01-01

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

  4. Cloning of a soluble isoform of the SgIGSF adhesion molecule that binds the extracellular domain of the membrane-bound isoform.

    OpenAIRE

    Koma, Yu-ichiro; Ito, Akihiko; Wakayama, Tomohiko; Watabe, Kenji; OKADA, Morihito; Tsubota, Noriaki; Iseki, Shoichi; Kitamura, Yukihiko

    2004-01-01

    SgIGSF (spermatogenic immunoglobulin superfamily) is a recently identified intercellular adhesion molecule of the immunoglobulin superfamily. In a mast-cell cDNA library, we found a clone that resulted from the retention of intron 7 within the mature SgIGSF message. This clone was predicted to encode a soluble isoform of SgIGSF (sSgIGSF) with 336 amino-acid residues because its open reading frame ended just before the transmembrane domain. We constructed a plasmid expressing sSgIGSF fused to ...

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

    Science.gov (United States)

    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

  6. Conditional expression of CD44 isoforms in lymphoma cells: influence on hyaluronate binding and tumor growth

    International Nuclear Information System (INIS)

    CD44 describes a family of surface proteins consisting of many isoforms due to alternative splice of ten 'variant' exons. Members of this family are involved in various processes including hematopoiesis, lymphocyte activation and homing, limb development, wound healing and tumor progression. Clinically, CD44 has been shown to be a prognostic factor for several human cancers. To answer the question which isoform might be relevant for tumor progression and to gain an insight into the mechanism of its function, I established transfectants of the LB lymphoma cell line in which the expression of four CD44 isoforms, namely CD44v3-10, CD44v4-10, CD44v8-10 and CD44s, was controlled by the Tet-off promoter. In the presence of Doxycycline, the expression was repressed. Removal of Doxycycline switched on expression and the maximal CD44 amount was obtained within two days. The transfectants were characterized regarding their ability to bind to the extracellular matrix component hyaluronate (HA). Overexpression of all four CD44 isoforms conferred the ability to bind HA on LB cells. Other glycosaminoglycans (GAGs) were bound in an isotype-specific fashion. CD44v3-10, CD44v4-10 and CD44v8-10 showed high binding affinity to chondroitin A, B and C, and low affinity to heparin, heparan sulfate and keratan sulfate. CD44s could not bind to these GAGs. Among these three variants, the binding ability of CD44v3-10 was the strongest. CD44 clustering seemed to play a crucial role for HA binding. Both CD44s and CD44v8-10 formed reduction-sensitive complexes in LB cells. The complexes are homooligomers or heterooligomers composed of different isoforms. Cys286 in CD44 transmember domain was not responsible for the formation of reduction-sensitive oligomer or for the enhanced HA binding in LB cell line. Using a conditional dimerization system the requirement of CD44 oligomerization for HA binding was directly demonstrated. The induction of oligomerization increased HA binding. Finally, I

  7. Conditional expression of CD44 isoforms in lymphoma cells: influence on hyaluronate binding and tumor growth

    Energy Technology Data Exchange (ETDEWEB)

    Fu, J.

    2002-03-01

    CD44 describes a family of surface proteins consisting of many isoforms due to alternative splice of ten 'variant' exons. Members of this family are involved in various processes including hematopoiesis, lymphocyte activation and homing, limb development, wound healing and tumor progression. Clinically, CD44 has been shown to be a prognostic factor for several human cancers. To answer the question which isoform might be relevant for tumor progression and to gain an insight into the mechanism of its function, I established transfectants of the LB lymphoma cell line in which the expression of four CD44 isoforms, namely CD44v3-10, CD44v4-10, CD44v8-10 and CD44s, was controlled by the Tet-off promoter. In the presence of Doxycycline, the expression was repressed. Removal of Doxycycline switched on expression and the maximal CD44 amount was obtained within two days. The transfectants were characterized regarding their ability to bind to the extracellular matrix component hyaluronate (HA). Overexpression of all four CD44 isoforms conferred the ability to bind HA on LB cells. Other glycosaminoglycans (GAGs) were bound in an isotype-specific fashion. CD44v3-10, CD44v4-10 and CD44v8-10 showed high binding affinity to chondroitin A, B and C, and low affinity to heparin, heparan sulfate and keratan sulfate. CD44s could not bind to these GAGs. Among these three variants, the binding ability of CD44v3-10 was the strongest. CD44 clustering seemed to play a crucial role for HA binding. Both CD44s and CD44v8-10 formed reduction-sensitive complexes in LB cells. The complexes are homooligomers or heterooligomers composed of different isoforms. Cys286 in CD44 transmember domain was not responsible for the formation of reduction-sensitive oligomer or for the enhanced HA binding in LB cell line. Using a conditional dimerization system the requirement of CD44 oligomerization for HA binding was directly demonstrated. The induction of oligomerization increased HA binding

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

    Directory of Open Access Journals (Sweden)

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

    OpenAIRE

    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...

  10. Multiple tissue-specific isoforms of sulfatide activate CD1d-restricted type II NKT cells

    DEFF Research Database (Denmark)

    Blomqvist, Maria; Rhost, Sara; Teneberg, Susann;

    2009-01-01

    relevant isoforms C24:1 and C24:0, major constituents of the myelin sheet of the nervous system, and C16:0, prominent in the pancreatic islet beta-cells. The most potent sulfatide isoform was lysosulfatide (lacking a fatty acid). Shortened fatty acid chain length (C24:1 versus C18:1), or saturation...... isoforms by a CD1d-restricted NKT-cell clone, and suggest that sulfatide, a major component of the myelin sheet and pancreatic beta-cells, is one of several natural ligands for type II CD1d-restricted NKT cells....

  11. The expression of ELK transcription factors in adult DRG: novel isoforms, antisense transcripts and upregulation by nerve damage

    OpenAIRE

    Kerr, Niall; Pintzas, Alexander; Holmes, Fiona; Hobson, Sally-Ann; Pope, Robert; Wallace, Mark; Wasylyk, Christine; Wasylyk, Bohdan; Wynick, David

    2010-01-01

    ELK transcription factors are expressed in brain, but it is unknown whether they are expressed in the peripheral nervous system. We show by RT-PCR that the previously described Elk1, Elk3/Elk3b/Elk3c and Elk4 mRNAs are expressed in adult dorsal root ganglia (DRG), together with the novel alternatively spliced isoforms Elk1b, Elk3d and Elk4c/Elk4d/Elk4e. These isoforms are also expressed in brain, heart, kidney and testis. In contrast to Elk3 protein, the novel Elk3d isoform is cytoplasmic, fa...

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

    Science.gov (United States)

    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

  13. A crustin isoform from black tiger shrimp, Penaeus monodon exhibits broad spectrum anti-bacterial activity

    Directory of Open Access Journals (Sweden)

    Debashis Banerjee

    2015-11-01

    Full Text Available Crustaceans have a powerful non-specific immune mechanism that responds to pathogen invasion and together with cellular responses, generates powerful humoral factors such as antimicrobial peptides. Crustins are a diverse class of antimicrobial peptides that are expressed by the circulating haemocytes of crustaceans. Several isoforms of this molecule are reported and in this study, one isoform from the black tiger shrimp, Penaeus monodon was cloned and expressed in Escherichia coli SG 13009. The purified recombinant crustin peptide had a molecular weight of 22 kDa and exhibited potent anti-bacterial activity in vitro against several Gram positive and Gram negative bacteria that included pathogens of aquatic animals and humans. The recombinant crustin showed a minimal inhibitory concentration of 0.5 μg ml−1 against the vibrio pathogens of shrimp, which suggests its promise for application in aquaculture.

  14. Signalling by Transforming Growth Factor Beta Isoforms in Wound Healing and Tissue Regeneration

    Directory of Open Access Journals (Sweden)

    Richard W.D. Gilbert

    2016-06-01

    Full Text Available Transforming growth factor beta (TGFβ signalling is essential for wound healing, including both non-specific scar formation and tissue-specific regeneration. Specific TGFβ isoforms and downstream mediators of canonical and non-canonical signalling play different roles in each of these processes. Here we review the role of TGFβ signalling during tissue repair, with a particular focus on the prototypic isoforms TGFβ1, TGFβ2, and TGFβ3. We begin by introducing TGFβ signalling and then discuss the role of these growth factors and their key downstream signalling mediators in determining the balance between scar formation and tissue regeneration. Next we discuss examples of the pleiotropic roles of TGFβ ligands during cutaneous wound healing and blastema-mediated regeneration, and how inhibition of the canonical signalling pathway (using small molecule inhibitors blocks regeneration. Finally, we review various TGFβ-targeting therapeutic strategies that hold promise for enhancing tissue repair.

  15. Detection of Diverse and High Molecular Weight Nesprin-1 and Nesprin-2 Isoforms Using Western Blotting.

    Science.gov (United States)

    Carthew, James; Karakesisoglou, Iakowos

    2016-01-01

    Heavily utilized in cell and molecular biology, western blotting is considered a crucial technique for the detection and quantification of proteins within complex mixtures. In particular, the detection of members of the nesprin (nuclear envelope spectrin repeat protein) family has proven difficult to analyze due to their substantial isoform diversity, molecular weight variation, and the sheer size of both nesprin-1 and nesprin-2 giant protein variants (>800 kDa). Nesprin isoforms contain distinct domain signatures, perform differential cytoskeletal associations, occupy different subcellular compartments, and vary in their tissue expression profiles. This structural and functional variance highlights the need to distinguish between the full range of proteins within the nesprin protein family, allowing for greater understanding of their specific roles in cell biology and disease. Herein, we describe a western blotting protocol modified for the detection of low to high molecular weight (50-1000 kDa) nesprin proteins. PMID:27147045

  16. Characterisation of human fibroblasts as keratinocyte feeder layer using p63 isoforms status.

    Science.gov (United States)

    Auxenfans, Céline; Thépot, Amélie; Justin, Virginie; Hautefeuille, Agnès; Shahabeddin, Lili; Damour, Odile; Hainaut, Pierre

    2009-01-01

    Large-scale culture of primary keratinocytes allows the production of large epidermal sheet surfaces for the treatment of extensive skin burns. This method is dependent upon the capacity to establish cultures of proliferating keratinocytes in conditions compatible with their clonal expansion while maintaining their capacity to differentiate into the typical squamous pattern of human epidermis. Feeder layers are critical in this process because the fibroblasts that compose this layer serve as a source of adhesion, growth and differentiation factors. In this report, we have characterise the expression patterns of p63 isoforms in primary keratinocytes cultured on two different feeder layer systems, murine 3T3 and human fibroblasts. We show that with the latter, keratinocytes express a higher ratio of Delta N to TAp63 isoform, in relation with higher clonogenic potential. These results indicate that human fibroblasts represent an adequate feeder layer system to support the culture of primary human keratinocytes. PMID:20042803

  17. Quarternary structure and enzymological properties of the different hormone-sensitive lipase (HSL) isoforms

    DEFF Research Database (Denmark)

    Krintel, Christian; Klint, Cecilia; Lindvall, Håkan;

    2010-01-01

    Hormone-sensitive lipase (HSL) is a key enzyme in the mobilization of energy in the form of fatty acids from intracellular stores of neutral lipids. The enzyme has been shown to exist in different isoforms with different molecular masses (84 kDa, 89 kDa and 117 kDa) expressed in a tissue-dependen......Hormone-sensitive lipase (HSL) is a key enzyme in the mobilization of energy in the form of fatty acids from intracellular stores of neutral lipids. The enzyme has been shown to exist in different isoforms with different molecular masses (84 kDa, 89 kDa and 117 kDa) expressed in a tissue...

  18. Metabolism of 7-benzyloxy-4-trifluoromethyl-coumarin by human hepatic cytochrome P450 isoforms.

    Science.gov (United States)

    Renwick, A B; Surry, D; Price, R J; Lake, B G; Evans, D C

    2000-10-01

    1. The metabolism of 7-benzyloxy-4-trifluoromethylcoumarin (BFC) to 7-hydroxy-4-trifluoromethylcoumarin (HFC) was studied in human liver microsomal preparations and in cDNA-expressed human cytochrome P450 (CYP) isoforms. 2. Kinetic analysis of the NADPH-dependent metabolism of BFC to HFC in four preparations of pooled human liver microsomes revealed mean (+/- SEM) Km and Vmax of 8.3 +/- 1.3 microM and 454 +/- 98 pmol/min/mg protein respectively. 3. The metabolism of BFC to HFC was determined in a characterized bank of 24 individual human liver microsomal preparations employing BFC substrate concentrations of 20 and 50 microM (i.e. about two and six times Km respectively). With 20 microM BFC the highest correlations were observed between BFC metabolism and markers of CYP1A2 (r2 = 0.784-0.797) and then with CYP3A (r2 = 0.434-0.547) isoforms, whereas with 50 microM BFC the highest correlations were observed between BFC metabolism and markers of CYP3A (r2 = 0.679-0.837) and then with CYP1A2 (r2 = 0.421-0.427) isoforms. At both BFC substrate concentrations, lower correlations were observed between BFC metabolism and enzymatic markers for CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP4A9/11. 4. Using human beta-lymphoblastoid cell microsomes containing cDNA-expressed CYP isoforms, 20 microM BFC was metabolized by CYP1A2 and CYP3A4, with lower rates of metabolism being observed with CYP2C9 and CYP2C19. Kinetic studies with the CYP1A2 and CYP3A4 preparations demonstrated a lower Km with the CYP1A2 preparation, but a higher Vmax with the CYP3A4 preparation. 5. The metabolism of 20 microM BFC in human liver microsomes was inhibited to 37-48% of control by 5-100 microM of the mechanism-based CYP1A2 inhibitor furafylline and to 64-69% of control by 5-100 microM of the mechanism-based CYP3A4 inhibitor troleandomycin. While some inhibition of BFC metabolism was observed in the presence of 100 and 200 microM diethyldithiocarbamate, the addition of 2-50 micro

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

    Directory of Open Access Journals (Sweden)

    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.

  20. Alternatively spliced short and long isoforms of adaptor protein intersectin 1 form complexes in mammalian cells

    Directory of Open Access Journals (Sweden)

    Rynditch A. V.

    2012-12-01

    Full Text Available Intersectin 1 (ITSN1 is an adaptor protein involved in membrane trafficking and cell signaling. Long and short isoforms of ITSN1 (ITSN1-L and ITSN1-S are produced by alternative splicing. The aim of our study was to investigate whether ITSN1-L and ITSN1-S could interact in mammalian cells. Methods. During this study we employed immunoprecipitation and confocal microscopy. Results. We have shown that endogenous ITSN1-S co-precipitates with overexpressed ITSN1-L in PC12, 293 and 293T cells. Long and short isoforms of ITSN1 also co-localize in 293T cells. Conclusions. ITSN1-L and ITSN1-S form complexes in mammalian cells.

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

    Science.gov (United States)

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

    2016-02-25

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

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

    DEFF Research Database (Denmark)

    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...

  3. Isoforms of agrin are widely expressed in the developing rat and may function as protease inhibitors.

    Science.gov (United States)

    Biroc, S L; Payan, D G; Fisher, J M

    1993-09-17

    The agrin family of extracellular matrix proteins may be important in the formation of the neuromuscular junction. Using in situ hybridization with a probe recognizing all agrin isoforms, we demonstrate that it is widely expressed during mammalian embryogenesis. In the developing rat, particularly high levels of expression are found in the dorsal root and cranial ganglia, gut, whisker rudiments, penis, snout, teeth, retina, hippocampus, cerebral cortex and the lining of brain ventricles. Functional analysis of the recombinant rat protein shows that it is a potent inhibitor of the proteases trypsin, chymotrypsin and plasmin but not thrombin or the plasminogen activators. We conclude that agrin and its isoforms may play multiple roles in mammalian development including the regulation of proteolysis in the extracellular matrix.

  4. Effects of eugenol on T-type Ca2+ channel isoforms.

    Science.gov (United States)

    Seo, Haengsoo; Li, Hai Ying; Perez-Reyes, Edward; Lee, Jung-Ha

    2013-11-01

    Eugenol has been used as an analgesic in dentistry. Previous studies have demonstrated that voltage-gated Na(+) channels and high-voltage-activated Ca(2+) channels expressed in trigeminal ganglion (TG) neurons sensing dental pain are molecular targets of eugenol for its analgesic effects. However, it has not been investigated whether eugenol can affect T-type Ca(2+) channels, which are known to be detected in the afferent neurons. In this report, we investigate how eugenol can influence cloned T-type channel isoforms expressed in HEK293 cells, using whole-cell patch clamp. Application of eugenol inhibited Cav3.1, Cav3.2, and Cav3.3 currents in a concentration-dependent manner with IC50 values of 463, 486, and 708 μM, respectively. Eugenol was found to negatively shift the steady-state inactivation curves of the T-type channel isoforms, but it did not shift their activation curves. In addition, eugenol had little effect on the current kinetics of Cav3.1 and Cav3.2, but it accelerated the inactivation kinetics of Cav3.3 currents. Reduction of channel availability enhanced eugenol inhibition sensitivity for Cav3.1 and Cav3.2, but not for Cav3.3. Moreover, eugenol inhibition of T-type channel isoforms was found to be use dependent. Finally, we show that the T-type currents recorded from rat TG neurons were inhibited by eugenol with a similar potency to Cav3.1 and Cav3.2 isoforms. Taken together, our findings suggest that T-type Ca(2+) channels are additional molecular targets for the pain-relieving effects of eugenol.

  5. PSD-95 mediates membrane clustering of the human plasma membrane Ca2+ pump isoform 4b

    OpenAIRE

    Padányi, Rita; Pászty, Katalin; STREHLER, EMANUEL E.; Enyedi, Ágnes

    2008-01-01

    Besides the control of global calcium changes, specific plasma membrane calcium ATPase (PMCA) isoforms are involved in the regulation of local calcium signals. Although local calcium signaling requires the confinement of signaling molecules into microdomains, little is known about the specific organization of PMCA molecules within the plasma membrane. Here we show that co-expression with the postsynaptic–density-95 (PSD-95) scaffolding protein increased the plasma membrane expression of PMCA4...

  6. Improved mass spectrometry assay for plasma hepcidin: detection and characterization of a novel hepcidin isoform.

    Directory of Open Access Journals (Sweden)

    Coby M M Laarakkers

    Full Text Available Mass spectrometry (MS-based assays for the quantification of the iron regulatory hormone hepcidin are pivotal to discriminate between the bioactive 25-amino acid form that can effectively block the sole iron transporter ferroportin and other naturally occurring smaller isoforms without a known role in iron metabolism. Here we describe the design, validation and use of a novel stable hepcidin-25(+40 isotope as internal standard for quantification. Importantly, the relative large mass shift of 40 Da makes this isotope also suitable for easy-to-use medium resolution linear time-of-flight (TOF platforms. As expected, implementation of hepcidin-25(+40 as internal standard in our weak cation exchange (WCX TOF MS method yielded very low inter/intra run coefficients of variation. Surprisingly, however, in samples from kidney disease patients, we detected a novel peak (m/z 2673.9 with low intensity that could be identified as hepcidin-24 and had previously remained unnoticed due to peak interference with the formerly used internal standard. Using a cell-based bioassay it was shown that synthetic hepcidin-24 was, like the -22 and -20 isoforms, a significantly less potent inducer of ferroportin degradation than hepcidin-25. During prolonged storage of plasma at room temperature, we observed that a decrease in plasma hepcidin-25 was paralleled by an increase in the levels of the hepcidin-24, -22 and -20 isoforms. This provides first evidence that all determinants for the conversion of hepcidin-25 to smaller inactive isoforms are present in the circulation, which may contribute to the functional suppression of hepcidin-25, that is significantly elevated in patients with renal impairment. The present update of our hepcidin TOF MS assay together with improved insights in the source and preparation of the internal standard, and sample stability will further improve our understanding of circulating hepcidin and pave the way towards further optimization and

  7. Two farnesoid X receptor alpha isoforms in Japanese medaka (Oryzias latipes) are differentially activated in vitro

    International Nuclear Information System (INIS)

    The nuclear receptor farnesoid X receptor alpha (FXRα, NR1H4) is activated by bile acids in multiple species including mouse, rat, and human and in this study we have identified two isoforms of Fxrα in Japanese medaka (Oryzias latipes), a small freshwater teleost. Both isoforms share a high amino acid sequence identity to mammalian FXRα (∼70% in the ligand-binding domain). Fxrα1 and Fxrα2 differ within the AF1 domain due to alternative splicing at the fourth intron-exon boundary. This process results in Fxrα1 having an extended N-terminus compared to Fxrα2. A Gal4DBD-FxrαLBD fusion construct was activated by chenodeoxycholic, cholic, deoxycholic and lithocholic acids, and the synthetic agonist GW4064 in transient transactivation assays. Activation of the Gal4DBD-FxrαLBD fusion construct was enhanced by addition of PGC-1α, as demonstrated through titration assays. Surprisingly, when the full-length versions of the two Fxrα isoforms were compared in transient transfection assays, Fxrα2 was activated by C24 bile acids and GW4064, while Fxrα1 was not significantly activated by any of the compounds tested. Since the only significant difference between the full-length constructs was sequence in the AF1 domain, these experiments highlight a key functional region in the Fxrα AF1 domain. Furthermore, mammalian two-hybrid studies demonstrated the ability of Fxrα2, but not Fxrα1, to interact with PGC-1α and SRC-1, and supported our results from the transient transfection reporter gene activation assays. These data demonstrate that both mammalian and teleost FXR (Fxrα2 isoform) are activated by primary and secondary bile acids.

  8. Two farnesoid X receptor alpha isoforms in Japanese medaka (Oryzias latipes) are differentially activated in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Howarth, Deanna L. [Integrated Toxicology and Environmental Health Program and Nicholas School of the Environment and Earth Sciences, Duke University, Durham, NC 27708 (United States); Hagey, Lee R. [Department of Medicine, University of California at San Diego, La Jolla, CA 92093 (United States); Law, Sheran H.W. [Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC 27695 (United States); Ai, Ni [Department of Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854 (United States); Krasowski, Matthew D. [Department of Pathology, University of Iowa Hospitals and Clinics, Iowa City, IA (United States); Ekins, Sean [Department of Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854 (United States); Collaboration in Chemistry, Jenkintown, PA 19046 (United States); Department of Pharmaceutical Sciences, University of Maryland, Baltimore, MD 21201 (United States); Moore, John T. [GlaxoSmithKline Discovery Research, Research Triangle Park, NC 27709 (United States); Kollitz, Erin M. [Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC 27695 (United States); Hinton, David E. [Integrated Toxicology and Environmental Health Program and Nicholas School of the Environment and Earth Sciences, Duke University, Durham, NC 27708 (United States); Kullman, Seth W., E-mail: swkullma@ncsu.edu [Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC 27695 (United States)

    2010-07-01

    The nuclear receptor farnesoid X receptor alpha (FXR{alpha}, NR1H4) is activated by bile acids in multiple species including mouse, rat, and human and in this study we have identified two isoforms of Fxr{alpha} in Japanese medaka (Oryzias latipes), a small freshwater teleost. Both isoforms share a high amino acid sequence identity to mammalian FXR{alpha} ({approx}70% in the ligand-binding domain). Fxr{alpha}1 and Fxr{alpha}2 differ within the AF1 domain due to alternative splicing at the fourth intron-exon boundary. This process results in Fxr{alpha}1 having an extended N-terminus compared to Fxr{alpha}2. A Gal4DBD-Fxr{alpha}LBD fusion construct was activated by chenodeoxycholic, cholic, deoxycholic and lithocholic acids, and the synthetic agonist GW4064 in transient transactivation assays. Activation of the Gal4DBD-Fxr{alpha}LBD fusion construct was enhanced by addition of PGC-1{alpha}, as demonstrated through titration assays. Surprisingly, when the full-length versions of the two Fxr{alpha} isoforms were compared in transient transfection assays, Fxr{alpha}2 was activated by C{sub 24} bile acids and GW4064, while Fxr{alpha}1 was not significantly activated by any of the compounds tested. Since the only significant difference between the full-length constructs was sequence in the AF1 domain, these experiments highlight a key functional region in the Fxr{alpha} AF1 domain. Furthermore, mammalian two-hybrid studies demonstrated the ability of Fxr{alpha}2, but not Fxr{alpha}1, to interact with PGC-1{alpha} and SRC-1, and supported our results from the transient transfection reporter gene activation assays. These data demonstrate that both mammalian and teleost FXR (Fxr{alpha}2 isoform) are activated by primary and secondary bile acids.

  9. iReckon: Simultaneous isoform discovery and abundance estimation from RNA-seq data

    OpenAIRE

    Mezlini, Aziz M.; Smith, Eric J. M.; Fiume, Marc; Buske, Orion; Savich, Gleb L.; Shah, Sohrab; Aparicio, Sam; Chiang, Derek Y.; Goldenberg, Anna; Brudno, Michael

    2013-01-01

    High-throughput RNA sequencing (RNA-seq) promises to revolutionize our understanding of genes and their role in human disease by characterizing the RNA content of tissues and cells. The realization of this promise, however, is conditional on the development of effective computational methods for the identification and quantification of transcripts from incomplete and noisy data. In this article, we introduce iReckon, a method for simultaneous determination of the isoforms and estimation of th...

  10. Revealing the functions of the transketolase enzyme isoforms in Rhodopseudomonas palustris using a systems biology approach.

    Directory of Open Access Journals (Sweden)

    Chia-Wei Hu

    Full Text Available BACKGROUND: Rhodopseudomonas palustris (R. palustris is a purple non-sulfur anoxygenic phototrophic bacterium that belongs to the class of proteobacteria. It is capable of absorbing atmospheric carbon dioxide and converting it to biomass via the process of photosynthesis and the Calvin-Benson-Bassham (CBB cycle. Transketolase is a key enzyme involved in the CBB cycle. Here, we reveal the functions of transketolase isoforms I and II in R. palustris using a systems biology approach. METHODOLOGY/PRINCIPAL FINDINGS: By measuring growth ability, we found that transketolase could enhance the autotrophic growth and biomass production of R. palustris. Microarray and real-time quantitative PCR revealed that transketolase isoforms I and II were involved in different carbon metabolic pathways. In addition, immunogold staining demonstrated that the two transketolase isoforms had different spatial localizations: transketolase I was primarily associated with the intracytoplasmic membrane (ICM but transketolase II was mostly distributed in the cytoplasm. Comparative proteomic analysis and network construction of transketolase over-expression and negative control (NC strains revealed that protein folding, transcriptional regulation, amino acid transport and CBB cycle-associated carbon metabolism were enriched in the transketolase I over-expressed strain. In contrast, ATP synthesis, carbohydrate transport, glycolysis-associated carbon metabolism and CBB cycle-associated carbon metabolism were enriched in the transketolase II over-expressed strain. Furthermore, ATP synthesis assays showed a significant increase in ATP synthesis in the transketolase II over-expressed strain. A PEPCK activity assay showed that PEPCK activity was higher in transketolase over-expressed strains than in the negative control strain. CONCLUSIONS/SIGNIFICANCE: Taken together, our results indicate that the two isoforms of transketolase in R. palustris could affect photoautotrophic growth

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

    OpenAIRE

    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...

  12. Decreased Levels of the Gelsolin Plasma Isoform in Patients with Rheumatoid Arthritis

    OpenAIRE

    Osborn, Teresia M.; Verdrengh, Margareta; Stossel, Thomas Peter; Tarkowski, Andrej; Bokarewa, Maria

    2008-01-01

    Introduction Gelsolin is an intracellular actin-binding protein involved in cell shape changes, cell motility, and apoptosis. An extracellular gelsolin isoform, plasma gelsolin circulates in the blood of healthy individuals at a concentration of \\(200 \\pm 50\\) mg/L and has been suggested to be a key component of an extracellular actin-scavenging system during tissue damage. Levels of plasma gelsolin decrease during acute injury and inflammation, and administration of recombinant plasma gelsol...

  13. New isoforms and assembly of glutamine synthetase in the leaf of wheat (Triticum aestivum L.).

    Science.gov (United States)

    Wang, Xiaochun; Wei, Yihao; Shi, Lanxin; Ma, Xinming; Theg, Steven M

    2015-11-01

    Glutamine synthetase (GS; EC 6.3.1.2) plays a crucial role in the assimilation and re-assimilation of ammonia derived from a wide variety of metabolic processes during plant growth and development. Here, three developmentally regulated isoforms of GS holoenzyme in the leaf of wheat (Triticum aestivum L.) seedlings are described using native-PAGE with a transferase activity assay. The isoforms showed different mobilities in gels, with GSII>GSIII>GSI. The cytosolic GSI was composed of three subunits, GS1, GSr1, and GSr2, with the same molecular weight (39.2kDa), but different pI values. GSI appeared at leaf emergence and was active throughout the leaf lifespan. GSII and GSIII, both located in the chloroplast, were each composed of a single 42.1kDa subunit with different pI values. GSII was active mainly in green leaves, while GSIII showed brief but higher activity in green leaves grown under field conditions. LC-MS/MS experiments revealed that GSII and GSIII have the same amino acid sequence, but GSII has more modification sites. With a modified blue native electrophoresis (BNE) technique and in-gel catalytic activity analysis, only two GS isoforms were observed: one cytosolic and one chloroplastic. Mass calibrations on BNE gels showed that the cytosolic GS1 holoenzyme was ~490kDa and likely a dodecamer, and the chloroplastic GS2 holoenzyme was ~240kDa and likely a hexamer. Our experimental data suggest that the activity of GS isoforms in wheat is regulated by subcellular localization, assembly, and modification to achieve their roles during plant development.

  14. Prokaryotic expression and characterization of a pea actin isoform (PEAcl) fused to GFP

    Institute of Scientific and Technical Information of China (English)

    ZHANG Shaobin; REN Dongtao; XU Xiaojing; LIU Guoqin

    2004-01-01

    Actins widely exist in eukaryotic cells and play important roles in many living activities. As there are many kinds of actin isoforms in plant cells, it is difficult to purify each actin isoform in sufficient quantities for analyzing its physicochemical properties. In the present study, a pea(Pisum Sativum L.) actin isoform (PEAc1) fused to His-tag at its amino terminus and GFP (green fluorescent protein) at its Carboxyl terminus were expressed in E. Coli in inclusion bodies. The fusion protein (PEAc1-GFP) was highly purified with the yield of above 2 mg/L culture by dissolving inclusions in 8 mol/L urea, renaturing by dialysis in a gradient of urea, and affinity binding to Ni-resin. The purified mono meric PEAc1-GFP could efficiently bind on Dnase Ⅰ and inhibit the latter's enzyme activity. PEAc1-GFP could polymerize into green fluorescent filamentous structures (F-PEAc1-GFP), which could be labeled by TRITC-phalloidin, a specific agent for observing microfilaments. The PEAc1-GFP polymerization curve was identical with that of chicken skeletal muscle actin. The critical concentration for PEAc1-GFP to polymerize into filaments is 0.24 μmol/L. The F-PEAc1-GFP could stimulate myosin Mg-ATPase activity in a protein concentration dependant manner (about 4 folds at1 mg/mL F-PEAc1-GFP). The results above show that the PEAcl fused to GFP retained the assembly characteristic of actin, indicating that gene fusion, prokaryotic expression,denaturation and renaturation, and affinity chromatography is a useful strategy for obtaining plant actin isoform proteins in a large amount.

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

    OpenAIRE

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

    2015-01-01

    Background Starch-binding domains are key modules present in several enzymes involved in polysaccharide metabolism. These non-catalytic modules have already been described as essential for starch-binding and the catalytic activity of starch synthase III from the higher plant Arabidopsis thaliana. In Ostreococcus tauri, a unicellular green alga of the Prasinophyceae family, there are three SSIII isoforms, known as Ostta SSIII-A, SSIII-B and SSIII-C. Results In this work, using in silico and in...

  16. Cell elasticity is regulated by the tropomyosin isoform composition of the actin cytoskeleton.

    Science.gov (United States)

    Jalilian, Iman; Heu, Celine; Cheng, Hong; Freittag, Hannah; Desouza, Melissa; Stehn, Justine R; 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 a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments.

  17. Formation of VEGF isoform-specific spatial distributions governing angiogenesis: computational analysis

    Directory of Open Access Journals (Sweden)

    Mac Gabhann Feilim

    2011-05-01

    Full Text Available Abstract Background The spatial distribution of vascular endothelial growth factor A (VEGF is an important mediator of vascular patterning. Previous experimental studies in the mouse hindbrain and retina have suggested that VEGF alternative splicing, which controls the ability of VEGF to bind to heparan sulfate proteoglycans (HSPGs in the extracellular matrix (ECM, plays a key role in controlling VEGF diffusion and gradients in tissues. Conversely, proteolysis notably by matrix metalloproteinases (MMPs, plays a critical role in pathological situations by releasing matrix-sequestered VEGF and modulating angiogenesis. However, computational models have predicted that HSPG binding alone does not affect VEGF localization or gradients at steady state. Results Using a 3D molecular-detailed reaction-diffusion model of VEGF ligand-receptor kinetics and transport, we test alternate models of VEGF transport in the extracellular environment surrounding an endothelial sprout. We show that differences in localization between VEGF isoforms, as observed experimentally in the mouse hindbrain, as well as the ability of proteases to redistribute VEGF in pathological situations, are consistent with a model where VEGF is endogenously cleared or degraded in an isoform-specific manner. We use our predictions of the VEGF distribution to quantify a tip cell's receptor binding and gradient sensing capacity. A novel prediction is that neuropilin-1, despite functioning as a coreceptor to VEGF165-VEGFR2 binding, reduces the ability of a cell to gauge the relative steepness of the VEGF distribution. Comparing our model to available in vivo vascular patterning data suggests that vascular phenotypes are most consistently predicted at short range by the soluble fraction of the VEGF distributions, or at longer range by matrix-bound VEGF detected in a filopodia-dependent manner. Conclusions Isoform-specific VEGF degradation provides a possible explanation for numerous examples

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

    International Nuclear Information System (INIS)

    Highlights: •We discovered a new member of VEGFxxxb family-VEGF111b. •We found VEGF111b mRNA and protein can be induced by mitomycin C. •We confirmed VEGF111b over-expression inhibits angiogenesis. •VEGF111b inhibits angiogenesis through inhibiting VEGF-R2/PI3K/Akt and VEGF-R2/ERK1/2 phosphorylation. -- Abstract: Vascular endothelial growth factor (VEGF-A) stimulating angiogenesis is required for tumor growth and progression. The conventional VEGF-A isoforms have been considered as pro-angiogenic factors. Another family of VEGF-A isoforms generated by alternative splicing, termed VEGFxxxb isoforms, has anti-angiogenic property, exemplified by VEGF165b. Here, we identify a new number of VEGFxxx family-VEGF111b induced by mitomycin C, although not detected in mitomycin C-unexposed ovarian cancer cells. SKOV3 cells were transfected with pcDNA3.1 empty vector, pcDNA3.1-VEGF111b or pcDNA3.1-VEGF165b to collect conditioned mediums respectively. VEGF111b overexpression inhibits proliferation, migration and tube formation of endothelial cell by inhibiting VEGF-R2 phosphorylation and its downstream signaling, similar to VEGF165b but slightly lower than VEGF165b. The anti-angiogenic property depends on the six amino acids of exon 8b of the VEGFxxxb isoforms. Our results show that VEGF111b is a novel potent anti-angiogenic agent that can target the VEGF-R2 and its signaling pathway to inhibit ovarian tumor growth

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

    OpenAIRE

    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...

  20. Characterization of fusion genes and the significantly expressed fusion isoforms in breast cancer by hybrid sequencing

    OpenAIRE

    Weirather, Jason L.; Afshar, Pegah Tootoonchi; Clark, Tyson A.; Tseng, Elizabeth; Powers, Linda S.; Underwood, Jason G; Zabner, Joseph; Korlach, Jonas; Wong, Wing Hung; Au, Kin Fai

    2015-01-01

    We developed an innovative hybrid sequencing approach, IDP-fusion, to detect fusion genes, determine fusion sites and identify and quantify fusion isoforms. IDP-fusion is the first method to study gene fusion events by integrating Third Generation Sequencing long reads and Second Generation Sequencing short reads. We applied IDP-fusion to PacBio data and Illumina data from the MCF-7 breast cancer cells. Compared with the existing tools, IDP-fusion detects fusion genes at higher precision and ...

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

    Science.gov (United States)

    Eilers, Wouter; Gevers, Wouter; van Overbeek, Daniëlle; de Haan, Arnold; Jaspers, Richard T.; Hilbers, Peter A.; van Riel, Natal; Flück, Martin

    2014-01-01

    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. PMID:25054156

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

    Directory of Open Access Journals (Sweden)

    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.

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

    OpenAIRE

    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...

  4. 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

    Directory of Open Access Journals (Sweden)

    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.

  5. Alternative Splicing Generates Different Parkin Protein Isoforms: Evidences in Human, Rat, and Mouse Brain

    Directory of Open Access Journals (Sweden)

    Soraya Scuderi

    2014-01-01

    Full Text Available Parkinson protein 2, E3 ubiquitin protein ligase (PARK2 gene mutations are the most frequent causes of autosomal recessive early onset Parkinson’s disease and juvenile Parkinson disease. Parkin deficiency has also been linked to other human pathologies, for example, sporadic Parkinson disease, Alzheimer disease, autism, and cancer. PARK2 primary transcript undergoes an extensive alternative splicing, which enhances transcriptomic diversification. To date several PARK2 splice variants have been identified; however, the expression and distribution of parkin isoforms have not been deeply investigated yet. Here, the currently known PARK2 gene transcripts and relative predicted encoded proteins in human, rat, and mouse are reviewed. By analyzing the literature, we highlight the existing data showing the presence of multiple parkin isoforms in the brain. Their expression emerges from conflicting results regarding the electrophoretic mobility of the protein, but it is also assumed from discrepant observations on the cellular and tissue distribution of parkin. Although the characterization of each predicted isoforms is complex, since they often diverge only for few amino acids, analysis of their expression patterns in the brain might account for the different pathogenetic effects linked to PARK2 gene mutations.

  6. Isoform-specific regulation and localization of the coxsackie and adenovirus receptor in human airway epithelia.

    Directory of Open Access Journals (Sweden)

    Katherine J D A Excoffon

    Full Text Available Adenovirus is an important respiratory pathogen. Adenovirus fiber from most serotypes co-opts the Coxsackie-Adenovirus Receptor (CAR to bind and enter cells. However, CAR is a cell adhesion molecule localized on the basolateral membrane of polarized epithelia. Separation from the lumen of the airways by tight junctions renders airway epithelia resistant to inhaled adenovirus infection. Although a role for CAR in viral spread and egress has been established, the mechanism of initial respiratory infection remains controversial. CAR exists in several protein isoforms including two transmembrane isoforms that differ only at the carboxy-terminus (CAR(Ex7 and CAR(Ex8. We found low-level expression of the CAR(Ex8 isoform in well-differentiated human airway epithelia. Surprisingly, in contrast to CAR(Ex7, CAR(Ex8 localizes to the apical membrane of epithelia where it augments adenovirus infection. Interestingly, despite sharing a similar class of PDZ-binding domain with CAR(Ex7, CAR(Ex8 differentially interacts with PICK1, PSD-95, and MAGI-1b. MAGI-1b appears to stoichiometrically regulate the degradation of CAR(Ex8 providing a potential mechanism for the apical localization of CAR(Ex8 in airway epithelial. In summary, apical localization of CAR(Ex8 may be responsible for initiation of respiratory adenoviral infections and this localization appears to be regulated by interactions with PDZ-domain containing proteins.

  7. Effect of silencing the two major tomato fruit pectin methylesterase isoforms on cell wall pectin metabolism.

    Science.gov (United States)

    Wen, B; Ström, A; Tasker, A; West, G; Tucker, G A

    2013-11-01

    Post-harvest storage is largely limited by fruit softening, a result of cell wall degradation. Pectin methylesterase (PE) (EC 3.1.1.11) is a major hydrolase responsible for pectin de-esterification in the cell wall, a response to fruit ripening. Two major PE isoforms, PE1 and PE2, have been isolated from tomato (Solanum lycopersicon) pericarp tissue and both have previously been down-regulated using antisense suppression. In this paper, PE1 and PE2 double antisense tomato plants were successfully generated through crossing the two single antisense lines. In the double antisense fruit, approximately 10% of normal PE activity remained and ripening associated pectin de-esterification was almost completely blocked. However, double antisense fruit softened normally during ripening. In tomato fruit, the PE1 isoform was found to contribute little to total PE activity and have little effect on the degree of esterification of pectin. In contrast, the other dominant fruit isoform, PE2, has a major impact on de-esterification of total pectin. PE2 appears to act on non-CDTA-soluble pectin during ripening and on CDTA-soluble pectin before the start of ripening in a potentially block-wise fashion.

  8. Probing polypeptide GalNAc-transferase isoform substrate specificities by in vitro analysis.

    Science.gov (United States)

    Kong, Yun; Joshi, Hiren J; Schjoldager, Katrine Ter-Borch Gram; Madsen, Thomas Daugbjerg; Gerken, Thomas A; Vester-Christensen, Malene B; Wandall, Hans H; Bennett, Eric Paul; Levery, Steven B; Vakhrushev, Sergey Y; Clausen, Henrik

    2015-01-01

    N-acetylgalactosaminyltransferase (GalNAc)-type (mucin-type) O-glycosylation is an abundant and highly diverse modification of proteins. This type of O-glycosylation is initiated in the Golgi by a large family of up to 20 homologous polypeptide GalNAc-T isoenzymes that transfer GalNAc to Ser, Thr and possibly Tyr residues. These GalNAc residues are then further elongated by a large set of glycosyltransferases to build a variety of complex O-glycan structures. What determines O-glycan site occupancy is still poorly understood, although it is clear that the substrate specificities of individual isoenzymes and the repertoire of GalNAc-Ts in cells are key parameters. The GalNAc-T isoenzymes are differentially expressed in cells and tissues in principle allowing cells to produce unique O-glycoproteomes dependent on the specific subset of isoforms present. In vitro analysis of acceptor peptide substrate specificities using recombinant expressed GalNAc-Ts has been the method of choice for probing activities of individual isoforms, but these studies have been hampered by biological validation of actual O-glycosylation sites in proteins and number of substrate testable. Here, we present a systematic analysis of the activity of 10 human GalNAc-T isoenzymes with 195 peptide substrates covering known O-glycosylation sites and provide a comprehensive dataset for evaluating isoform-specific contributions to the O-glycoproteome.

  9. Peroxiredoxin isoforms are associated with cardiovascular risk factors in type 2 diabetes mellitus

    Energy Technology Data Exchange (ETDEWEB)

    El Eter, E. [Physiology Department, Faculty of Medicine, King Saud University, Riyadh (Saudi Arabia); Cardiovascular Research Group, Faculty of Medicine, King Saud University, Riyadh (Saudi Arabia); Physiology Department, Faculty of Medicine, Alexandria University, Alexandria (Egypt); Al-Masri, A.A. [Physiology Department, Faculty of Medicine, King Saud University, Riyadh (Saudi Arabia); Cardiovascular Research Group, Faculty of Medicine, King Saud University, Riyadh (Saudi Arabia)

    2015-03-03

    The production of oxygen free radicals in type 2 diabetes mellitus contributes to the development of complications, especially the cardiovascular-related ones. Peroxiredoxins (PRDXs) are antioxidant enzymes that combat oxidative stress. The aim of this study was to investigate the associations between the levels of PRDX isoforms (1, 2, 4, and 6) and cardiovascular risk factors in type 2 diabetes mellitus. Fifty-three patients with type 2 diabetes mellitus (28F/25M) and 25 healthy control subjects (7F/18M) were enrolled. We measured the plasma levels of each PRDX isoform and analyzed their correlations with cardiovascular risk factors. The plasma PRDX1, -2, -4, and -6 levels were higher in the diabetic patients than in the healthy control subjects. PRDX2 and -6 levels were negatively correlated with diastolic blood pressure, fasting blood sugar, and hemoglobin A1c. In contrast, PRDX1 levels were positively correlated with low-density lipoprotein and C-reactive protein levels. PRDX4 levels were negatively correlated with triglycerides. In conclusion, PRDX1, -2, -4, and -6 showed differential correlations with a variety of traditional cardiovascular risk factors. These results should encourage further research into the crosstalk between PRDX isoforms and cardiovascular risk factors.

  10. Energy-optimised pharmacophore approach to identify potential hotspots during inhibition of Class II HDAC isoforms.

    Science.gov (United States)

    Ganai, Shabir Ahmad; Shanmugam, Karthi; Mahadevan, Vijayalakshmi

    2015-01-01

    Histone deacetylases (HDACs) are conjugated enzymes that modulate chromatin architecture by deacetylating lysine residues on the histone tails leading to transcriptional repression. Pharmacological interventions of these enzymes with small molecule inhibitors called Histone deacetylase inhibitors (HDACi) have shown enhanced acetylation of the genome and are hence emerging as potential targets at the clinic. Type-specific inhibition of Class II HDACs has shown enhanced therapeutic benefits against developmental and neurodegenerative disorders. However, the structural identity of class-specific isoforms limits the potential of their inhibitors in precise targeting of their enzymes. Diverse strategies have been implemented to recognise the features in HDAC enzymes which may help in identifying isoform specificity factors. This work attempts a computational approach that combines in silico docking and energy-optimised pharmacophore (E-pharmacophore) mapping of 18 known HDAC inhibitors and has identified structural variations that regulate their interactions against the six Class II HDAC enzymes considered for the study. This combined approach establishes that inhibitors possessing higher number of aromatic rings in different structural regions might function as potent inhibitors, while inhibitors with scarce ring structures might point to compromised potency. This would aid the rationale for chemical optimisation and design of isoform selective HDAC inhibitors with enhanced affinity and therapeutic efficiency.

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

    Science.gov (United States)

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

    2005-09-01

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

  12. Role of p73 Dinucleotide Polymorphism in Prostate Cancer and p73 Protein Isoform Balance

    Directory of Open Access Journals (Sweden)

    L. Michael Carastro

    2014-01-01

    Full Text Available Background. Molecular markers for prostate cancer (PCa risks are currently lacking. Here we address the potential association of a dinucleotide polymorphism (DNP in exon 2 of the p73 gene with PCa risk/progression and discern any disruption of p73 protein isoforms levels in cells harboring a p73 DNP allele. Methods. We investigated the association between p73 DNP genotype and PCa risk/aggressiveness and survival by fitting logistic regression models in 1,292 incident cases and 682 controls. Results. Although we detected no association between p73 DNP and PCa risk, a significant inverse relationship between p73 DNP and PCa aggressiveness (AT/AT + GC/AT versus GC/GC, OR = 0.55, 95%Cl = 0.31–0.99 was detected. Also, p73 DNP is marginally associated with overall death (dominant model, HR = 0.76, 95%Cl = 0.57–1.00, P=0.053 as well as PCa specific death (HR = 0.69, 95%Cl = 0.45–1.06, P=0.09. Western blot analyses for p73 protein isoforms indicate that cells heterozygous for the p73 DNP have lower levels of ∆Np73 relative to TAp73 (P<0.001. Conclusions. Our findings are consistent with an association between p73 DNP and low risk for PCa aggressiveness by increasing the expressed TAp73/∆Np73 protein isoform ratio.

  13. Mechanisms controlling Pax6 isoform expression in the retina have been conserved between teleosts and mammals.

    Science.gov (United States)

    Lakowski, Jörn; Majumder, Anirban; Lauderdale, James D

    2007-07-15

    The Pax6 gene plays several roles in retinal development, including control of cell proliferation, maintenance of the retinogenic potential of progenitor cells, and cell fate specification. Emerging evidence suggests that these different aspects of Pax6 gene function are mediated by different isoforms of the Pax6 protein; however, relatively little is known about the spatiotemporal expression of Pax6 isoforms in the vertebrate retina. Using bacterial artificial chromosome (BAC) technology, we modified a zebrafish Pax6a BAC such that we could distinguish paired-containing Pax6a transcripts from paired-less Pax6a transcripts. In the zebrafish, the spatial and temporal onset of expression of these transcripts suggests that the paired-less isoform is involved in the cell fate decision leading to the generation of amacrine cells; however, because of limitations associated with transient transgenic analysis, it was not feasible to establish whether this promoter was active in all amacrine cells or in a specific population of amacrine cells. By making mice transgenic for the zebrafish Pax6a BAC reporter transgene, we were able to show that paired-containing and paired-less Pax6a transcripts were differentially expressed in amacrine subpopulations. Our study also directly demonstrates the functional conservation of the regulatory mechanisms governing Pax6 transcription in teleosts and mammals.

  14. Novel isoform of adaptor protein ITSN1 forms homodimers via its C-terminus

    Directory of Open Access Journals (Sweden)

    Skrypkina I. Ya.

    2011-06-01

    Full Text Available Aim. Previously we have identified a novel isoform of endocytic adaptor protein ITSN1 designated as ITSN122a. Western blot revealed two immunoreactive bands of 120 and 250 kDa that corresponded to ITSN1-22a. The goal of this study was to investigate the possibility of dimer formation by the novel isoform. Methods. Dimerization ability of ITSN1-22a was tested by immunoprecipitation and subsequent Western blot analysis. To specify the region responsible for dimerization, site-directed mutagenesis and truncation analysis were carried out. Inhibition of endocytosis by potassium depletion and EGF stimulation of HEK293 were performed. Results. We have found that ITSN1-22a forms dimers in HEK293 cells. The dimerization of ITSN1-22a was mediated by C-terminal domain. We showed that cysteines C1016 and C1019 were involved in homodimerization. Inhibition of clathrin-mediated endocytosis and mitogen stimulation did not affect ITSN1-22a dimer formation. Conclusions. ITSN1-22a is the only one known ITSN1 isoform, which is capable to form homodimers via disulphide bonds. This could be important for the formation of protein complexes containing ITSN1 molecules.

  15. Differential expression of two activating transcription factor 5 isoforms in papillary thyroid carcinoma

    Science.gov (United States)

    Vicari, Luisa; La Rosa, Cristina; Forte, Stefano; Calabrese, Giovanna; Colarossi, Cristina; Aiello, Eleonora; Salluzzo, Salvatore; Memeo, Lorenzo

    2016-01-01

    Background Activating transcription factor 5 (ATF5) is a member of the activating transcription/cAMP response element-binding protein family of basic leucine zipper proteins that plays an important role in cell survival, differentiation, proliferation, and apoptosis. The ATF5 gene generates two transcripts: ATF5 isoform 1 and ATF5 isoform 2. A number of studies indicate that ATF5 could be an attractive target for therapeutic intervention in several tumor types; however, so far, the role of ATF5 has not been investigated in papillary thyroid carcinoma (PTC). Methods Quantitative real-time reverse transcription polymerase chain reaction and immuno-histochemical staining were used to study ATF5 mRNA and protein expression in PTC. Results We report here that ATF5 is expressed more in PTC tissue than in normal thyroid tissue. Furthermore, this is the first study that describes the presence of both ATF5 isoforms in PTC. Conclusion These findings could provide potential applications in PTC cancer treatment.

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

    Science.gov (United States)

    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

  17. Temperature-dependent developmental variation in lobster muscle myosin heavy chain isoforms.

    Science.gov (United States)

    Magnay, J L; Holmes, J M; Neil, D M; El Haj, A J

    2003-10-16

    The temperature- and developmental-regulation of myosin heavy chain (MyHC) expression and primary sequence was investigated in the abdominal musculature of developing Homarus gammarus larvae acclimated to 10, 14 and 19+/-1 degrees C. MyHC loop 1 (ATP binding) and loop 2 (actin binding) regions were sequenced and compared. The deduced amino acid sequence of MyHC loop 1 showed a development-related increase in net charge from +1 to +2 between larval stages 1 and 2, which was not temperature-dependent. In post-settled stage 9 larvae, minor shifts in amino acid sequence occurred at 19 degrees C, and corresponded to a significant up-regulation of fast myosin mRNA expression. However, no temperature-specific loop 1 isoforms were detected. The deduced amino acid sequence of MyHC loop 2 was not affected by temperature, and the net charge remained +4 throughout development. These findings contrast to previous studies using the common carp, in which temperature-specific MyHC isoform genes were expressed in response to disparate thermal regimes. This raises the question as to whether arthropods do not express specific temperature isoforms but instead rely on shifts in fibre type to accommodate alterations in thermal environment. PMID:14563558

  18. Differential expression of a new isoform of DLG2 in renal oncocytoma

    Directory of Open Access Journals (Sweden)

    Kovacs Gyula

    2006-04-01

    Full Text Available Abstract Background Renal oncocytoma, a benign tumour of the kidney, may pose a differential diagnostic problem due to overlapping phenotype with chromophobe renal cell carcinoma or other types of renal cell tumours. Therefore, identification of molecular markers would be of great value for molecular diagnostics of this tumour type. Methods In the current study we applied various techniques, including Affymetrix microarray hybridization and semiquantitative RT-PCR, to identify genes expressed differentially in renal oncocytomas. Subsequently, we used RACE and Northern blot hybridization to characterize the potential candidates for molecular diagnosis. Results We have identified new isoform of DLG2 gene, which contains 3'-end exons of the known DLG2 gene along with the hypothetical gene FLJ37266. The new isoform is specifically upregulated in renal oncocytoma, whereas the known DLG2 gene is downregulated in this type of kidney tumour. Conclusion The new isoform of DLG2 is the promising candidate gene for molecular differential diagnostics of renal oncocytoma.

  19. Developmentally regulated switch in alternatively spliced SNAP-25 isoforms alters facilitation of synaptic transmission.

    Science.gov (United States)

    Bark, Christina; Bellinger, Frederick P; Kaushal, Ashutosh; Mathews, James R; Partridge, L Donald; Wilson, Michael C

    2004-10-01

    Although the basic molecular components that promote regulated neurotransmitter release are well established, the contribution of these proteins as regulators of the plasticity of neurotransmission and refinement of synaptic connectivity during development is elaborated less fully. For example, during the period of synaptic growth and maturation in brain, the expression of synaptosomal protein 25 kDa (SNAP-25), a neuronal t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) essential for action potential-dependent neuroexocytosis, is altered through alternative splicing of pre-mRNA transcripts. We addressed the role of the two splice-variant isoforms of SNAP-25 with a targeted mouse mutation that impairs the shift from SNAP-25a to SNAP-25b. Most of these mutant mice die between 3 and 5 weeks of age, which coincides with the time when SNAP-25b expression normally reaches mature levels in brain and synapse formation is essentially completed. The altered expression of these SNAP-25 isoforms influences short-term synaptic function by affecting facilitation but not the initial probability of release. This suggests that mechanisms controlling alternative splicing between SNAP-25 isoforms contribute to a molecular switch important for survival that helps to guide the transition from immature to mature synaptic connections, as well as synapse regrowth and remodeling after neural injury.

  20. Peroxiredoxin isoforms are associated with cardiovascular risk factors in type 2 diabetes mellitus

    International Nuclear Information System (INIS)

    The production of oxygen free radicals in type 2 diabetes mellitus contributes to the development of complications, especially the cardiovascular-related ones. Peroxiredoxins (PRDXs) are antioxidant enzymes that combat oxidative stress. The aim of this study was to investigate the associations between the levels of PRDX isoforms (1, 2, 4, and 6) and cardiovascular risk factors in type 2 diabetes mellitus. Fifty-three patients with type 2 diabetes mellitus (28F/25M) and 25 healthy control subjects (7F/18M) were enrolled. We measured the plasma levels of each PRDX isoform and analyzed their correlations with cardiovascular risk factors. The plasma PRDX1, -2, -4, and -6 levels were higher in the diabetic patients than in the healthy control subjects. PRDX2 and -6 levels were negatively correlated with diastolic blood pressure, fasting blood sugar, and hemoglobin A1c. In contrast, PRDX1 levels were positively correlated with low-density lipoprotein and C-reactive protein levels. PRDX4 levels were negatively correlated with triglycerides. In conclusion, PRDX1, -2, -4, and -6 showed differential correlations with a variety of traditional cardiovascular risk factors. These results should encourage further research into the crosstalk between PRDX isoforms and cardiovascular risk factors

  1. Human heterochromatin protein 1 isoforms regulate androgen receptor signaling in prostate cancer.

    Science.gov (United States)

    Itsumi, Momoe; Shiota, Masaki; Yokomizo, Akira; Kashiwagi, Eiji; Takeuchi, Ario; Tatsugami, Katsunori; Inokuchi, Junichi; Song, Yoohyun; Uchiumi, Takeshi; Naito, Seiji

    2013-06-01

    Androgen receptor (AR) signaling is critical for the tumorigenesis and development of prostate cancer, as well as the progression to castration-resistant prostate cancer. We previously showed that the heterochromatin protein 1 (HP1) β isoform plays a critical role in transactivation of AR signaling as an AR coactivator that promotes prostate cancer cell proliferation. However, the roles of other HP1 isoforms, HP1α and HP1γ, in AR expression and prostate cancer remain unclear. Here, we found that knockdown of HP1γ, but not HP1α, reduced AR expression and cell proliferation by inducing cell cycle arrest at G1 phase in LNCaP cells. Conversely, overexpression of full-length HP1α and its C-terminal deletion mutant increased AR expression and cell growth, whereas overexpression of HP1γ had no effect. Similarly, HP1α overexpression promoted 22Rv1 cell growth, whereas HP1γ knockdown reduced the proliferation of CxR cells, a castration-resistant LNCaP derivative. Taken together, HP1 isoforms distinctly augment AR signaling and cell growth in prostate cancer. Therefore, silencing of HP1β and HP1γ may be a promising therapeutic strategy for treatment of prostate cancer.

  2. Estrogen receptor isoforms and progestin hormone dependence in a mouse mammary tumor model.

    Science.gov (United States)

    Actis, A M; Caruso, S P; Levin, E

    1994-09-01

    The close interaction between receptors and other transcription factors suggests that their corresponding transducing signals can trigger functional and structural changes in other related molecules. The effect of a progestinic agent, medroxyprogesterone acetate (MPA), on some of the estrogen-receptor (ER) parameters was studied in 2 murine mammary tumor sublines with different progestin hormone dependence for their respective growth. The relative binding affinity of estradiol and tamoxifen for the ER, the receptor content and the ER isoforms studied by HPLC were determined in the hormone-autonomous (HA) and the hormone-dependent (HD) tumor sublines. In the HA subline administration of MPA did not modify the tumor growth rate, whereas this was accelerated in the HD subline. The ER content was clearly increased in the HD tumor subline, but not in the HA subline, compared with the untreated controls. In contrast, the E2 and tamoxifen relative binding affinity for the ER and the isoform profiles were affected by MPA treatment in the HA, but not in the HD tumor subline. The functional change (decrease in relative binding affinity) can be attributed to the appearance of a lower-molecular-size ER isoform under the progestinic treatment. Modifications in one receptor molecule by the action of ligands corresponding to another type of receptor show the interconection between transcription factors and the necessity of broadening conventional concepts regarding hormone dependence in mammary tumorigenesis. PMID:8077051

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2016-07-01

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

  5. Circadian rhythmicity of active GSK3 isoforms modulates molecular clock gene rhythms in the suprachiasmatic nucleus.

    Science.gov (United States)

    Besing, Rachel C; Paul, Jodi R; Hablitz, Lauren M; Rogers, Courtney O; Johnson, Russell L; Young, Martin E; Gamble, Karen L

    2015-04-01

    The suprachiasmatic nucleus (SCN) drives and synchronizes daily rhythms at the cellular level via transcriptional-translational feedback loops comprising clock genes such as Bmal1 and Period (Per). Glycogen synthase kinase 3 (GSK3), a serine/threonine kinase, phosphorylates at least 5 core clock proteins and shows diurnal variation in phosphorylation state (inactivation) of the GSK3β isoform. Whether phosphorylation of the other primary isoform (GSK3α) varies across the subjective day-night cycle is unknown. The purpose of this study was to determine if the endogenous rhythm of GSK3 (α and β) phosphorylation is critical for rhythmic BMAL1 expression and normal amplitude and periodicity of the molecular clock in the SCN. Significant circadian rhythmicity of phosphorylated GSK3 (α and β) was observed in the SCN from wild-type mice housed in constant darkness for 2 weeks. Importantly, chronic activation of both GSK3 isoforms impaired rhythmicity of the GSK3 target BMAL1. Furthermore, chronic pharmacological inhibition of GSK3 with 20 µM CHIR-99021 enhanced the amplitude and shortened the period of PER2::luciferase rhythms in organotypic SCN slice cultures. These results support the model that GSK3 activity status is regulated by the circadian clock and that GSK3 feeds back to regulate the molecular clock amplitude in the SCN.

  6. Changes in the expression of NO synthase isoforms after ozone: the effects of allergen exposure

    Directory of Open Access Journals (Sweden)

    Lee June-Hyuk

    2004-06-01

    Full Text Available Abstract Background The functional role of nitric oxide (NO and various nitric oxide synthase (NOS isoforms in asthma remains unclear. Objective This study investigated the effects of ozone and ovalbumin (OVA exposure on NOS isoforms. Methods The expression of inducible NOS (iNOS, neuronal NOS (nNOS, and endothelial NOS (eNOS in lung tissue was measured. Enhanced pause (Penh was measured as a marker of airway obstruction. Nitrate and nitrite in bronchoalveolar lavage (BAL fluid were measured using a modified Griess reaction. Results The nitrate concentration in BAL fluid from the OVA-sensitized/ozone-exposed/OVA-challenged group was greater than that of the OVA-sensitized/saline-challenged group. Methacholine-induced Penh was increased in the OVA-sensitized/ozone-exposed/OVA-challenged group, with a shift in the dose-response curve to the left, compared with the OVA-sensitized/saline-challenged group. The levels of nNOS and eNOS were increased significantly in the OVA-sensitized/ozone-exposed/OVA-challenged group and the iNOS levels were reduced compared with the OVA-sensitized/saline-challenged group. Conclusion In mice, ozone is associated with increases in lung eNOS and nNOS, and decreases in iNOS. None of these enzymes are further affected by allergens, suggesting that the NOS isoforms play different roles in airway inflammation after ozone exposure.

  7. Glutamine synthetase isoforms in nitrogen-fixing soybean nodules: distinct oligomeric structures and thiol-based regulation.

    Science.gov (United States)

    Masalkar, Pintu D; Roberts, Daniel M

    2015-01-16

    Legume root nodule glutamine synthetase (GS) catalyzes the assimilation of ammonia produced by nitrogen fixation. Two GS isoform subtypes (GS1β and GS1γ) are present in soybean nodules. GS1γ isoforms differ from GS1β isoforms in terms of their susceptibility to reversible inhibition by intersubunit disulfide bond formation between C159 and C92 at the shared active site at subunit interfaces. Although nodule GS enzymes share 86% amino acid sequence identity, analytical ultracentrifugation experiments showed that GS1γ is a dodecamer, whereas the GS1β is a decamer. It is proposed that this difference contributes to the differential thiol sensitivity of each isoform, and that GS1γ1 may be a target of thiol-based regulation.

  8. EXAFS analysis of a human Cu,Zn SOD isoform focused using non-denaturing gel electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Chevreux, Sylviane; Roudeau, Stephane; Deves, Guillaume; Ortega, Richard [Laboratoire de Chimie Nucleaire Analytique et Bioenvironnementale, CNRS UMR5084, Universite Bordeaux 1, Chemin du Solarium, F-33175 Gradignan cedex (France); Solari, Pier Lorenzo [Synchrotron SOLEIL, L' Orme des Merisiers, BP 48, F-91192 Gif-sur-Yvette cedex, Saint-Aubin (France); Alliot, Isabelle; Testemale, Denis; Hazemann, Jean Louis, E-mail: ortega@cenbg.in2p3.f [FAME, ESRF, 6 rue Jules Horowitz, BP220, F-38043 Grenoble cedex (France)

    2009-11-15

    Isoelectric point isoforms of a metalloprotein, copper-zinc superoxide dismutase (CuZnSOD), separated on electrophoresis gels were analyzed using X-ray Absorption Spectroscopy. Mutations of this protein are involved in familial cases of amyotrophic lateral sclerosis. The toxicity of mutants could be relied to defects in the metallation state. Our purpose is to establish analytical protocols to study metallation state of protein isoforms such as those from CuZnSOD. We previously highlighted differences in the copper oxidation state between CuZnSOD isoforms using XANES. Here, we present the first results for EXAFS analyses performed at Cu and Zn K-edge on the majoritary expressed isoform of human CuZnSOD separated on electrophoresis gels.

  9. EXAFS analysis of a human Cu,Zn SOD isoform focused using non-denaturing gel electrophoresis

    Science.gov (United States)

    Chevreux, Sylviane; Solari, Pier Lorenzo; Roudeau, Stéphane; Deves, Guillaume; Alliot, Isabelle; Testemale, Denis; Hazemann, Jean Louis; Ortega, Richard

    2009-11-01

    Isoelectric point isoforms of a metalloprotein, copper-zinc superoxide dismutase (CuZnSOD), separated on electrophoresis gels were analyzed using X-ray Absorption Spectroscopy. Mutations of this protein are involved in familial cases of amyotrophic lateral sclerosis. The toxicity of mutants could be relied to defects in the metallation state. Our purpose is to establish analytical protocols to study metallation state of protein isoforms such as those from CuZnSOD. We previously highlighted differences in the copper oxidation state between CuZnSOD isoforms using XANES. Here, we present the first results for EXAFS analyses performed at Cu and Zn K-edge on the majoritary expressed isoform of human CuZnSOD separated on electrophoresis gels.

  10. Activity of the acyl-CoA synthetase ACSL6 isoforms: role of the fatty acid Gate-domains

    Directory of Open Access Journals (Sweden)

    Siliakus Melvin

    2010-04-01

    Full Text Available Abstract Background Activation of fatty acids by acyl-CoA synthetase enzymes is required for de novo lipid synthesis, fatty acid catabolism, and remodeling of biological membranes. Human long-chain acyl-CoA synthetase member 6, ASCL6, is a form present in the plasma membrane of cells. Splicing events affecting the amino-terminus and alternative motifs near the ATP-binding site generate different isoforms of ACSL6. Results Isoforms with different fatty acid Gate-domain motifs have different activity and the form lacking this domain, isoform 3, showed no detectable activity. Enzymes truncated of the first 40 residues generate acyl-CoAs at a faster rate than the full-length protein. The gating residue, which prevents entry of the fatty acid substrate unless one molecule of ATP has already accessed the catalytic site, was identified as a tyrosine for isoform 1 and a phenylalanine for isoform 2 at position 319. All isoforms, with or without a fatty acid Gate-domain, as well as recombinant protein truncated of the N-terminus, can interact to form enzymatic complexes with identical or different isoforms. Conclusion The alternative fatty acid Gate-domain motifs are essential determinants for the activity of the human ACSL6 isoforms, which appear to act as homodimeric enzyme as well as in complex with other spliced forms. These findings provide evidence that the diversity of these enzyme species could produce the variety of acyl-CoA synthetase activities that are necessary to generate and repair the hundreds of lipid species present in membranes.

  11. Oligomeric properties and DNA binding specificities of repressor isoforms from the Streptomyces bacteriophage phiC31.

    Science.gov (United States)

    Wilson, S E; Smith, M C

    1998-05-15

    Three protein isoforms (74, 54 and 42 kDa) are expressed from repressor gene c in the Streptomyces temperate bacteriophage phiC31. Because expression of the two smaller isoforms, 54 and 42 kDa, is sufficient for superinfection immunity, the interaction between these isoforms was studied. The native 42 kDa repressor (Nat42) and an N-terminally 6x histidine-tagged 54 kDa isoform (His54) were shown by co-purification on a Ni-NTA column to interact in Streptomyces lividans . In vitro three repressor preparations, containing Nat42, His54 and the native 54 and 42 kDa isoforms expressed together (Nat54&42), were subjected to chemical crosslinking and gel filtration analysis. Homo- and hetero-tetramers were observed. Previous work showed that the smallest isoform bound to 17 bp operators containing aconservedinvertedrepeat (CIR) and that the CIRs were located at 16 loci throughout the phiC31 genome. One of the CIRs (CIR6) is believed to be critical for regulating the lytic pathway. The DNA binding activities of the three repressor preparations were studied using fragments containing CIRs (CIR3-CIR6) from the essential early region as templates for DNase I footprinting. Whereas Nat42 bound to CIR6, poorly to CIR5 but undetectably to CIR3 or CIR4, the Nat54&42 preparation could bind to all CIRs tested, albeit poorly to CIR3 and CIR4. The His54 isoform bound all CIRs tested. Isoforms expressed from the phiC31 repressor gene, like those which are expressed from many eukaryotic transcription factor genes, apparently have different binding specificities.

  12. Two Regions of the Tail Are Necessary for the Isoform-specific Functions of Nonmuscle Myosin IIB

    OpenAIRE

    Sato, Masaaki K.; Takahashi, Masayuki; Yazawa, Michio

    2007-01-01

    To function in the cell, nonmuscle myosin II molecules assemble into filaments through their C-terminal tails. Because myosin II isoforms most likely assemble into homo-filaments in vivo, it seems that some self-recognition mechanisms of individual myosin II isoforms should exist. Exogenous expression of myosin IIB rod fragment is thus expected to prevent the function of myosin IIB specifically. We expected to reveal some self-recognition sites of myosin IIB from the phenotype by expressing a...

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

    OpenAIRE

    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...

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

    OpenAIRE

    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...

  15. In Vitro Inhibitory Activities of the Extract of Hibiscus Sabdariffa L. (Family Malvaceae) on Selected Cytochrome P450 Isoforms

    OpenAIRE

    Johnson, Showande Segun; Oyelola, Fakeye Titilayo; Ari, Tolonen; Juho, Hokkanen

    2013-01-01

    Literature is scanty on the interaction potential of Hibiscus sabdariffa L., plant extract with other drugs and the affected targets. This study was conducted to investigate the cytochrome P450 (CYP) isoforms that are inhibited by the extract of Hibiscus sabdariffa L. in vitro. The inhibition towards the major drug metabolizing CYP isoforms by the plant extract were estimated in human liver microsomal incubations, by monitoring the CYP-specific model reactions through previously validated N-i...

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

    Science.gov (United States)

    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

  17. Loss of desmoplakin isoform I causes early onset cardiomyopathy and heart failure in a Naxos‐like syndrome

    Science.gov (United States)

    Uzumcu, A; Norgett, E E; Dindar, A; Uyguner, O; Nisli, K; Kayserili, H; Sahin, S E; Dupont, E; Severs, N J; Leigh, I M; Yuksel‐Apak, M; Kelsell, D P; Wollnik, B

    2006-01-01

    Background Desmosomes are cellular junctions important for intercellular adhesion and anchoring the intermediate filament (IF) cytoskeleton to the cell membrane. Desmoplakin (DSP) is the most abundant desmosomal protein with 2 isoforms produced by alternative splicing. Methods We describe a patient with a recessively inherited arrhythmogenic dilated cardiomyopathy with left and right ventricular involvement, epidermolytic palmoplantar keratoderma, and woolly hair. The patient showed a severe heart phenotype with an early onset and rapid progression to heart failure at 4 years of age. Results A homozygous nonsense mutation, R1267X, was found in exon 23 of the desmoplakin gene, which results in an isoform specific truncation of the larger DSPI isoform. The loss of most of the DSPI specific rod domain and C‐terminal area was confirmed by Western blotting and immunofluorescence. We further showed that the truncated DSPI transcript is unstable, leading to a loss of DSPI. DSPI is reported to be an obligate constituent of desmosomes and the only isoform present in cardiac tissue. To address this, we reviewed the expression of DSP isoforms in the heart. Our data suggest that DSPI is the major cardiac isoform but we also show that specific compartments of the heart have detectable DSPII expression. Conclusions This is the first description of a phenotype caused by a mutation affecting only one DSP isoform. Our findings emphasise the importance of desmoplakin and desmosomes in epidermal and cardiac function and additionally highlight the possibility that the different isoforms of desmoplakin may have distinct functional properties within the desmosome. PMID:16467215

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

    OpenAIRE

    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 ...

  19. Immunopositivity for histone macroH2A1 isoforms marks steatosis-associated hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Francesca Rappa

    Full Text Available BACKGROUND: Hepatocellular carcinoma (HCC is one of the most common cancers worldwide. Prevention and risk reduction are important and the identification of specific biomarkers for early diagnosis of HCC represents an active field of research. Increasing evidence indicates that fat accumulation in the liver, defined as hepatosteatosis, is an independent and strong risk factor for developing an HCC. MacroH2A1, a histone protein generally associated with the repressed regions of chromosomes, is involved in hepatic lipid metabolism and is present in two alternative spliced isoforms, macroH2A1.1 and macroH2A1.2. These isoforms have been shown to predict lung and colon cancer recurrence but to our knowledge, their role in fatty-liver associated HCC has not been investigated previously. METHODS: We examined macroH2A1.1 and macroH2A1.2 protein expression levels in the liver of two murine models of fat-associated HCC, the high fat diet/diethylnistrosamine (DEN and the phosphatase and tensin homolog (PTEN liver specific knock-out (KO mouse, and in human liver samples of subjects with steatosis or HCC, using immunoblotting and immunohistochemistry. RESULTS: Protein levels for both macroH2A1 isoforms were massively upregulated in HCC, whereas macroH2A1.2 was specifically upregulated in steatosis. In addition, examination of human liver samples showed a significant difference (p<0.01 in number of positive nuclei in HCC (100% of tumor cells positive for either macroH2A1.1 or macroH2A1.2, when compared to steatosis (<2% of hepatocytes positive for either isoform. The steatotic areas flanking the tumors were highly immunopositive for macroH2A1.1 and macroH2A1.2. CONCLUSIONS: These data obtained in mice and humans suggest that both macroH2A1 isoforms may play a role in HCC pathogenesis and moreover may be considered as novel diagnostic markers for human HCC.

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

    Science.gov (United States)

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

    2011-05-10

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-07-25

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

  2. Interaction of meropenem with 'N' and 'B' isoforms of human serum albumin: a spectroscopic and molecular docking study.

    Science.gov (United States)

    Rehman, Md Tabish; Ahmed, Sarfraz; Khan, Asad U

    2016-09-01

    Carbapenems are used to control the outbreak of β-lactamases expressing bacteria. The effectiveness of drugs is influenced by its interaction with human serum albumin (HSA). Strong binding of carbapenems to HSA may lead to decreased bioavailability of the drug. The non-optimal drug dosage will provide a positive selection pressure on bacteria to develop resistance. Here, we investigated the interaction between meropenem and HSA at physiological pH 7.5 (N-isoform HSA) and non-physiological pH 9.2 (B-isoform HSA). Results showed that meropenem quenches the fluorescence of both 'N' and 'B' isoforms of HSA (ΔG < 0 and binding constant ~10(4) M(-1)). Electrostatic interactions and van der Waal interactions along with H-bonds stabilized the complex of meropenem with 'N' and 'B' isoforms of HSA, respectively. Molecular docking results revealed that meropenem binds to HSA near Sudlow's site II (subdomain IIIA) close to Trp-214 with a contribution of a few residues of subdomain IIA. CD spectroscopy showed a change in the conformation of both the isoforms of HSA upon meropenem binding. The catalytic efficiency of HSA (only N-isoform) on p-nitrophenyl acetate was increased primarily due to a decrease in Km and an increase in kcat values. This study provides an insight into the molecular basis of interaction between meropenem and HSA.

  3. The Regulation of Nitric Oxide Synthase Isoform Expression in Mouse and Human Fallopian Tubes: Potential Insights for Ectopic Pregnancy

    Directory of Open Access Journals (Sweden)

    Junting Hu

    2014-12-01

    Full Text Available Nitric oxide (NO is highly unstable and has a half-life of seconds in buffer solutions. It is synthesized by NO-synthase (NOS, which has been found to exist in the following three isoforms: neuro nitric oxide synthase (nNOS, inducible nitric oxide synthase (iNOS, and endothelial nitric oxide synthase (eNOS. NOS activity is localized in the reproductive tracts of many species, although direct evidence for NOS isoforms in the Fallopian tubes of mice is still lacking. In the present study, we investigated the expression and regulation of NOS isoforms in the mouse and human Fallopian tubes during the estrous and menstrual cycles, respectively. We also measured isoform expression in humans with ectopic pregnancy and in mice treated with lipopolysaccharide (LPS. Our results confirmed the presence of different NOS isoforms in the mouse and human Fallopian tubes during different stages of the estrous and menstrual cycles and showed that iNOS expression increased in the Fallopian tubes of women with ectopic pregnancy and in LPS-treated mice. Elevated iNOS activity might influence ovulation, cilia beats, contractility, and embryo transportation in such a manner as to increase the risk of ectopic pregnancy. This study has provided morphological and molecular evidence that NOS isoforms are present and active in the human and mouse Fallopian tubes and suggests that iNOS might play an important role in both the reproductive cycle and infection-induced ectopic pregnancies.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    1989-01-01

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

  6. VEGF-A isoform-specific regulation of calcium ion flux, transcriptional activation and endothelial cell migration

    Directory of Open Access Journals (Sweden)

    Gareth W. Fearnley

    2015-07-01

    Full Text Available Vascular endothelial growth factor A (VEGF-A regulates many aspects of vascular physiology such as cell migration, proliferation, tubulogenesis and cell-cell interactions. Numerous isoforms of VEGF-A exist but their physiological significance is unclear. Here we evaluated two different VEGF-A isoforms and discovered differential regulation of cytosolic calcium ion flux, transcription factor localisation and endothelial cell response. Analysis of VEGF-A isoform-specific stimulation of VEGFR2-dependent signal transduction revealed differential capabilities for isoform activation of multiple signal transduction pathways. VEGF-A165 treatment promoted increased phospholipase Cγ1 phosphorylation, which was proportional to the subsequent rise in cytosolic calcium ions, in comparison to cells treated with VEGF-A121. A major consequence of this VEGF-A isoform-specific calcium ion flux in endothelial cells is differential dephosphorylation and subsequent nuclear translocation of the transcription factor NFATc2. Using reverse genetics, we discovered that NFATc2 is functionally required for VEGF-A-stimulated endothelial cell migration but not tubulogenesis. This work presents a new mechanism for understanding how VEGF-A isoforms program complex cellular outputs by converting signal transduction pathways into transcription factor redistribution to the nucleus, as well as defining a novel role for NFATc2 in regulating the endothelial cell response.

  7. Interaction of meropenem with 'N' and 'B' isoforms of human serum albumin: a spectroscopic and molecular docking study.

    Science.gov (United States)

    Rehman, Md Tabish; Ahmed, Sarfraz; Khan, Asad U

    2016-09-01

    Carbapenems are used to control the outbreak of β-lactamases expressing bacteria. The effectiveness of drugs is influenced by its interaction with human serum albumin (HSA). Strong binding of carbapenems to HSA may lead to decreased bioavailability of the drug. The non-optimal drug dosage will provide a positive selection pressure on bacteria to develop resistance. Here, we investigated the interaction between meropenem and HSA at physiological pH 7.5 (N-isoform HSA) and non-physiological pH 9.2 (B-isoform HSA). Results showed that meropenem quenches the fluorescence of both 'N' and 'B' isoforms of HSA (ΔG < 0 and binding constant ~10(4) M(-1)). Electrostatic interactions and van der Waal interactions along with H-bonds stabilized the complex of meropenem with 'N' and 'B' isoforms of HSA, respectively. Molecular docking results revealed that meropenem binds to HSA near Sudlow's site II (subdomain IIIA) close to Trp-214 with a contribution of a few residues of subdomain IIA. CD spectroscopy showed a change in the conformation of both the isoforms of HSA upon meropenem binding. The catalytic efficiency of HSA (only N-isoform) on p-nitrophenyl acetate was increased primarily due to a decrease in Km and an increase in kcat values. This study provides an insight into the molecular basis of interaction between meropenem and HSA. PMID:26372227

  8. Identification of T-cell factor-4 isoforms that contribute to the malignant phenotype of hepatocellular carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Tsedensodnom, Orkhontuya [Liver Research Center, Rhode Island Hospital and The Warren Alpert Medical School of Brown University, Providence, RI (United States); Department of Molecular Biology Cell Biology and Biochemistry, The Warren Alpert Medical School of Brown University, Providence, RI (United States); Koga, Hironori; Rosenberg, Stephen A.; Nambotin, Sarah B.; Carroll, John J.; Wands, Jack R. [Liver Research Center, Rhode Island Hospital and The Warren Alpert Medical School of Brown University, Providence, RI (United States); Kim, Miran, E-mail: Miran_Kim@brown.edu [Liver Research Center, Rhode Island Hospital and The Warren Alpert Medical School of Brown University, Providence, RI (United States)

    2011-04-15

    The Wnt/{beta}-catenin signaling pathway is frequently activated in hepatocellular carcinoma (HCC). Downstream signaling events involving the Wnt/{beta}-catenin cascade occur through T-cell factor (TCF) proteins. The human TCF-4 gene is composed of 17 exons with multiple alternative splicing sites. However, the role of different TCF-4 isoforms in the pathogenesis of HCC is unknown. The purpose of this study was to identify and characterize TCF-4 isoforms in HCC. We identified 14 novel TCF-4 isoforms from four HCC cell lines. Functional analysis following transfection and expression in HCC cells revealed distinct effects on the phenotype. The TCF-4J isoform expression produced striking features of malignant transformation characterized by high cell proliferation rate, migration and colony formation even though its transcriptional activity was low. In contrast, the TCF-4K isoform displayed low TCF transcriptional activity; cell proliferation rate and colony formation were reduced as well. Interestingly, TCF-4J and TCF-4K differed by only five amino acids (the SxxSS motif). Thus, these studies suggest that conserved splicing motifs may have a major influence on the transcriptional activity and functional properties of TCF-4 isoforms and alter the characteristics of the malignant phenotype.

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

    Science.gov (United States)

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

    2009-03-01

    A visually evoked signalling cascade, which begins in the retina, transverses the choroid, and mediates scleral remodelling, is considered to control eye growth. The ubiquitous cytokine TGF-beta has been associated with alterations in ocular growth, where alterations in scleral TGF-beta isoforms mediate the scleral remodelling that results in myopia. However, while the TGF-beta isoforms have been implicated in the scleral change during myopia development, it is unclear whether alterations in retinal and choroidal isoforms constitute part of the retinoscleral cascade. This study characterised the retinal and choroidal TGF-beta isoform profiles and TGF-beta2 activation during different stages of myopia development, as induced by form deprivation, in a mammalian model of eye growth. Using quantitative real-time PCR, the mRNA for all three mammalian isoforms of TGF-beta was detected in tree shrew retina and choroid. Distinct tissue-specific isoform profiles were observed for the retina (TGF-beta1:TGF-beta2:TGF-beta3=20:2085:1) and choroid (TGF-beta1:TGF-beta2:TGF-beta3=16:23:1), which remained constant over the development period under investigation. The active and latent pools of retinal TGF-beta2 were quantified using ELISA with the majority (>94%) of total TGF-beta2 found in the latent form. Unlike previous scleral data showing early and continuous decreases in TGF-beta isoform expression during myopia development, the levels of the three isoforms remained within normal ranges for retinal (TGF-beta1, -14 to +14%; TGF-beta2, -2 to +20%; TGF-beta3, -10 to +26%) and choroidal (TGF-beta1, -19 to +21%; TGF-beta2, -26 to +8%; TGF-beta3, -11 to +28%) tissues during myopia development (induction times of 3h, 7h, 11h, 24h, and 5 days). A 40% decrease in retinal TGF-beta2 activation was observed after 5 days of myopia development, however, there was no functional correlate of altered TGF-beta2 activity, as assessed by the retinal ERG response. Overall, these data highlight

  10. CD44 isoforms are heterogeneously expressed in breast cancer and correlate with tumor subtypes and cancer stem cell markers

    International Nuclear Information System (INIS)

    The CD44 cell adhesion molecule is aberrantly expressed in many breast tumors and has been implicated in the metastatic process as well as in the putative cancer stem cell (CSC) compartment. We aimed to investigate potential associations between alternatively spliced isoforms of CD44 and CSCs as well as to various breast cancer biomarkers and molecular subtypes. We used q-RT-PCR and exon-exon spanning assays to analyze the expression of four alternatively spliced CD44 isoforms as well as the total expression of CD44 in 187 breast tumors and 13 cell lines. ALDH1 protein expression was determined by IHC on TMA. Breast cancer cell lines showed a heterogeneous expression pattern of the CD44 isoforms, which shifted considerably when cells were grown as mammospheres. Tumors characterized as positive for the CD44+/CD24- phenotype by immunohistochemistry were associated to all isoforms except the CD44 standard (CD44S) isoform, which lacks all variant exons. Conversely, tumors with strong expression of the CSC marker ALDH1 had elevated expression of CD44S. A high expression of the CD44v2-v10 isoform, which retain all variant exons, was correlated to positive steroid receptor status, low proliferation and luminal A subtype. The CD44v3-v10 isoform showed similar correlations, while high expression of CD44v8-v10 was correlated to positive EGFR, negative/low HER2 status and basal-like subtype. High expression of CD44S was associated with strong HER2 staining and also a subgroup of basal-like tumors. Unsupervised hierarchical cluster analysis of CD44 isoform expression data divided tumors into four main clusters, which showed significant correlations to molecular subtypes and differences in 10-year overall survival. We demonstrate that individual CD44 isoforms can be associated to different breast cancer subtypes and clinical markers such as HER2, ER and PgR, which suggests involvement of CD44 splice variants in specific oncogenic signaling pathways. Efforts to link CD44 to CSCs

  11. First Trimester Pregnancy Loss and the Expression of alternatively spliced NKp30 isoforms in Maternal Blood and Placental Tissue

    Directory of Open Access Journals (Sweden)

    Avishai eShemesh

    2015-06-01

    Full Text Available In this study, we aimed to investigate whether first trimester pregnancy loss is associated with differences in expression of NKp30 splice variants (isoforms in maternal peripheral blood or placental tissue. We conducted a prospective case-control study; a total of 33 women undergoing dilation and curettage due to first trimester pregnancy loss were further subdivided into groups with sporadic or recurrent pregnancy loss. The control group was comprised of women undergoing elective termination of pregnancy. The qPCR approach was employed to assess the relative expression of NKp30 isoforms as well as the total expression of NKp30 and NKp46 receptors between the selected groups. Results show that in both PBMC and placental tissue, NKp46 and NKp30 expression was mildly elevated in the pregnancy loss groups compared with the elective group. In particular, NKp46 elevation was significant. Moreover, expression analysis of NKp30 isoforms manifested a different profile between PBMC and the placenta. NKp30-a and NKp30-b isoforms in the placental tissue, but not in PBMC, showed a significant increase in the pregnancy loss groups compared with the elective group. Placental expression of NKp30 activating isoforms -a and -b in the pregnancy loss groups was negatively correlated with PLGF expression. In contrast, placental expression of these isoforms in the elective group was positively correlated with TNFα, IL-10 and VEGF-A expression. The altered expression of NKp30 activating isoforms in placental tissue from patients with pregnancy loss compared to the elective group and the different correlations with cytokine expression point to the involvement of NKp30-mediated function in pregnancy loss.

  12. Transcript Isoform Variation Associated with Cytosine Modification in Human Lymphoblastoid Cell Lines.

    Science.gov (United States)

    Zhang, Xu; Zhang, Wei

    2016-06-01

    Cytosine modification on DNA is variable among individuals, which could correlate with gene expression variation. The effect of cytosine modification on interindividual transcript isoform variation (TIV), however, remains unclear. In this study, we assessed the extent of cytosine modification-specific TIV in lymphoblastoid cell lines (LCLs) derived from unrelated individuals of European and African descent. Our study detected cytosine modification-specific TIVs for 17% of the analyzed genes at a 5% false discovery rate. Forty-five percent of the TIV-associated cytosine modifications correlated with the overall gene expression levels as well, with the corresponding CpG sites overrepresented in transcript initiation sites, transcription factor binding sites, and distinct histone modification peaks, suggesting that alternative isoform transcription underlies the TIVs. Our analysis also revealed 33% of the TIV-associated cytosine modifications that affected specific exons, with the corresponding CpG sites overrepresented in exon/intron junctions, splicing branching points, and transcript termination sites, implying that the TIVs are attributable to alternative splicing or transcription termination. Genetic and epigenetic regulation of TIV shared target preference but exerted independent effects on 61% of the common exon targets. Cytosine modification-specific TIVs detected from LCLs were differentially enriched in those detected from various tissues in The Cancer Genome Atlas, indicating their developmental dependency. Genes containing cytosine modification-specific TIVs were enriched in pathways of cancers and metabolic disorders. Our study demonstrated a prominent effect of cytosine modification variation on the transcript isoform spectrum over gross transcript abundance and revealed epigenetic contributions to diseases that were mediated through cytosine modification-specific TIV. PMID:27029734

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

    Directory of Open Access Journals (Sweden)

    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.

  14. Protein kinase d isoforms differentially modulate cofilin-driven directed cell migration.

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    Heike Döppler

    Full Text Available BACKGROUND: Protein kinase D (PKD enzymes regulate cofilin-driven actin reorganization and directed cell migration through both p21-activated kinase 4 (PAK4 and the phosphatase slingshot 1L (SSH1L. The relative contributions of different endogenous PKD isoforms to both signaling pathways have not been elucidated, sufficiently. METHODOLOGY/PRINCIPAL FINDINGS: We here analyzed two cell lines (HeLa and MDA-MB-468 that express the subtypes protein kinase D2 (PKD2 and protein kinase D3 (PKD3. We show that under normal growth conditions both isoforms can form a complex, in which PKD3 is basally-active and PKD2 is inactive. Basal activity of PKD3 mediates PAK4 activity and downstream signaling, but does not significantly inhibit SSH1L. This signaling constellation was required for facilitating directed cell migration. Activation of PKD2 and further increase of PKD3 activity leads to additional phosphorylation and inhibition of endogenous SSH1L. Net effect is a dramatic increase in phospho-cofilin and a decrease in cell migration, since now both PAK4 and SSH1L are regulated by the active PKD2/PKD3 complex. CONCLUSIONS/SIGNIFICANCE: Our data suggest that PKD complexes provide an interface for both cofilin regulatory pathways. Dependent on the activity of involved PKD enzymes signaling can be balanced to guarantee a functional cofilin activity cycle and increase cell migration, or imbalanced to decrease cell migration. Our data also provide an explanation of how PKD isoforms mediate different effects on directed cell migration.

  15. Contribution of NADH increases to ethanol’s inhibition of retinol oxidation by human ADH isoforms

    Science.gov (United States)

    Chase, Jennifer R.; Poolman, Mark G.; Fell, David A.

    2010-01-01

    Background A decrease in retinoic acid levels due to alcohol consumption has been proposed as a contributor to such conditions as fetal alcohol spectrum diseases and ethanol-induced cancers. One molecular mechanism, competitive inhibition by ethanol of the catalytic activity of human alcohol dehydrogenase (EC 1.1.1.1) (ADH) on all-trans retinol oxidation has been shown for the ADH7 isoform. Ethanol metabolism also causes an increase in the free NADH in cells, which might reasonably be expected to decrease the retinol oxidation rate by product inhibition of ADH isoforms. Method To understand the relative importance of these two mechanisms by which ethanol decreases the retinol oxidation in vivo we need to assess them quantitatively. We have built a model system of four reactions: (1) ADH oxidation of ethanol and NAD+ (2) ADH oxidation of retinol and NAD+ (3) oxidation of ethanol by a generalized Ethanoloxidase that uses NAD+ (4) NADHoxidase which carries out NADH turnover. Results Using the metabolic modeling package SCRUMPY, we have shown that the ethanol-induced increase in NADH contributes from 0–90% of the inhibition by ethanol, depending on [ethanol] and ADH isoform. Furthermore, while the majority of flux control of retinaldehyde production is exerted by ADH, Ethanoloxidase and the NADHoxidase contribute as well. Discussion Our results show that the ethanol-induced increase in NADH makes a contribution of comparable importance to the ethanol competitive inhibition throughout the range of conditions likely to occur in vivo, and must be considered in the assessment of the in vivo mechanism of ethanol interference with fetal development and other diseases. PMID:19183134

  16. Distinct apolipoprotein E isoform preference for inhibition of smooth muscle cell migration and proliferation.

    Science.gov (United States)

    Zeleny, Michelle; Swertfeger, Debi K; Weisgraber, Karl H; Hui, David Y

    2002-10-01

    The current study compared the effectiveness of the various human apolipoprotein E (apoE) isoforms in inhibiting platelet-derived growth factor- (PDGF-) stimulated smooth muscle cell proliferation and migration. The incubation of primary mouse aortic smooth muscle cells with apoE3 resulted in dose-dependent inhibition of smooth muscle cells stimulated by 10 ng/mL PDGF. Greater than 50% inhibition of smooth muscle cell proliferation was observed at 15 microg/mL of human apoE3. Human apoE2 was less effective, requiring a higher concentration to achieve inhibition comparable to that of apoE3. Human apoE4 was the least effective of the apoE isoforms with no significant inhibition of cell proliferation observed at concentrations up to 15 microg/mL. Interestingly, apoE inhibition of PDGF-directed smooth muscle cell migration did not show preference for any apoE isoforms. Human apoE2, apoE3, and apoE4 were equally effective in inhibiting smooth muscle cell migration toward PDGF. These results are consistent with previous data showing that apoE inhibition of smooth muscle cell proliferation is mediated through its binding to heparan sulfate proteoglycans, whereas its inhibition of cell migration is mediated via binding to the low-density lipoprotein receptor related protein. The low efficiency of apoE4 to inhibit smooth muscle cell proliferation also suggested another mechanism to explain the association between the apolipoprotein epsilon4 allele with increased risk of coronary artery disease. PMID:12269825

  17. A novel alternatively spliced isoform of the mu-opioid receptor: functional antagonism

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    Wentworth Sean

    2010-06-01

    Full Text Available Abstract Background Opioids are the most widely used analgesics for the treatment of clinical pain. They produce their therapeutic effects by binding to μ-opioid receptors (MORs, which are 7 transmembrane domain (7TM G-protein-coupled receptors (GPCRs, and inhibiting cellular activity. However, the analgesic efficacy of opioids is compromised by side-effects such as analgesic tolerance, dependence and opioid-induced hyperalgesia (OIH. In contrast to opioid analgesia these side effects are associated with cellular excitation. Several hypotheses have been advanced to explain these phenomena, yet the molecular mechanisms underlying tolerance and OIH remain poorly understood. Results We recently discovered a new human alternatively spliced isoform of MOR (MOR1K that is missing the N-terminal extracellular and first transmembrane domains, resulting in a 6TM GPCR variant. To characterize the pattern of cellular transduction pathways activated by this human MOR1K isoform, we conducted a series of pharmacological and molecular experiments. Results show that stimulation of MOR1K with morphine leads to excitatory cellular effects. In contrast to stimulation of MOR1, stimulation of MOR1K leads to increased Ca2+ levels as well as increased nitric oxide (NO release. Immunoprecipitation experiments further reveal that unlike MOR1, which couples to the inhibitory Gαi/o complex, MOR1K couples to the stimulatory Gαs complex. Conclusion The major MOR1 and the alternative MOR1K isoforms mediate opposite cellular effects in response to morphine, with MOR1K driving excitatory processes. These findings warrant further investigations that examine animal and human MORK1 expression and function following chronic exposure to opioids, which may identify MOR1K as a novel target for the development of new clinically effective classes of opioids that have high analgesic efficacy with diminished ability to produce tolerance, OIH, and other unwanted side-effects.

  18. Somatodendritic and excitatory postsynaptic distribution of neuron-type dystrophin isoform, Dp40, in hippocampal neurons

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    Fujimoto, Takahiro; Itoh, Kyoko, E-mail: kxi14@koto.kpu-m.ac.jp; Yaoi, Takeshi; Fushiki, Shinji

    2014-09-12

    Highlights: • Identification of dystrophin (Dp) shortest isoform, Dp40, is a neuron-type Dp. • Dp40 expression is temporally and differentially regulated in comparison to Dp71. • Somatodendritic and nuclear localization of Dp40. • Dp40 is localized to excitatory postsynapses. • Dp40 might play roles in dendritic and synaptic functions. - Abstract: The Duchenne muscular dystrophy (DMD) gene produces multiple dystrophin (Dp) products due to the presence of several promoters. We previously reported the existence of a novel short isoform of Dp, Dp40, in adult mouse brain. However, the exact biochemical expression profile and cytological distribution of the Dp40 protein remain unknown. In this study, we generated a polyclonal antibody against the NH{sub 2}-terminal region of the Dp40 and identified the expression profile of Dp40 in the mouse brain. Through an analysis using embryonic and postnatal mouse cerebrums, we found that Dp40 emerged from the early neonatal stages until adulthood, whereas Dp71, an another Dp short isoform, was highly detected in both prenatal and postnatal cerebrums. Intriguingly, relative expressions of Dp40 and Dp71 were prominent in cultured dissociated neurons and non-neuronal cells derived from mouse hippocampus, respectively. Furthermore, the immunocytological distribution of Dp40 was analyzed in dissociated cultured neurons, revealing that Dp40 is detected in the soma and its dendrites, but not in the axon. It is worthy to note that Dp40 is localized along the subplasmalemmal region of the dendritic shafts, as well as at excitatory postsynaptic sites. Thus, Dp40 was identified as a neuron-type Dp possibly involving dendritic and synaptic functions.

  19. Adaptive evolution and elucidating the potential inhibitor against schizophrenia to target DAOA (G72 isoforms

    Directory of Open Access Journals (Sweden)

    Sehgal SA

    2015-07-01

    Full Text Available Sheikh Arslan Sehgal,1,2 Shazia Mannan,2,* Sumaira Kanwal,2,* Ishrat Naveed,1 Asif Mir1 1Department of Bioinformatics and Biotechnology, International Islamic University, Islamabad, Pakistan; 2Department of Biosciences, COMSATS Institute of Information Technology, Sahiwal, Pakistan *These authors contributed equally to this work Abstract: Schizophrenia (SZ, a chronic mental and heritable disorder characterized by neurophysiological impairment and neuropsychological abnormalities, is strongly associated with d-amino acid oxidase activator (DAOA, G72. Research studies emphasized that overexpression of DAOA may be responsible for improper functioning of neurotransmitters, resulting in neurological disorders like SZ. In the present study, a hybrid approach of comparative modeling and molecular docking followed by inhibitor identification and structure modeling was employed. Screening was performed by two-dimensional similarity search against selected inhibitor, keeping in view the physiochemical properties of the inhibitor. Here, we report an inhibitor compound which showed maximum binding affinity against four selected isoforms of DAOA. Docking studies revealed that Glu-53, Thr-54, Lys-58, Val-85, Ser-86, Tyr-87, Leu-88, Glu-90, Leu-95, Val-98, Ser-100, Glu-112, Tyr-116, Lys-120, Asp-121, and Arg-122 are critical residues for receptor–ligand interaction. The C-terminal of selected isoforms is conserved, and binding was observed on the conserved region of isoforms. We propose that selected inhibitor might be more potent on the basis of binding energy values. Further analysis of this inhibitor through site-directed mutagenesis could be helpful for exploring the details of ligand-binding pockets. Overall, the findings of this study may be helpful in designing novel therapeutic targets to cure SZ. Keywords: schizophrenia, bioinformatics, modeling, docking, DAOA, G72, DAO, computer-aided drug designing, phylogenetic analysis, d-amino acid oxidase

  20. 14-3-3 phosphoprotein interaction networks – does isoform diversity present functional interaction specification?

    Directory of Open Access Journals (Sweden)

    Anna-Lisa ePaul

    2012-08-01

    Full Text Available The 14-3-3 proteins have emerged as major phosphoprotein interaction proteins and thereby constitute a key node in the Arabidopsis Interactome Map, a node through which a large number of important signals pass. Throughout their history of discovery and description, the 14-3-3s have been described as protein families and there has been some evidence that the different 14-3-3 family members within any organisms might carry isoform specific functions. However there has also been evidence for redundancy of 14-3-3 function, suggesting that the perceived 14-3-3 diversity may be the accumulation of neutral mutations over evolutionary time and as some 14-3-3 genes develop tissue or organ specific expression. This situation has led to a currently unresolved question – does 14-3-3 isoform sequence diversity indicate functional diversity at the biochemical or cellular level? We discuss here some of the key observations on both sides of the resulting debate, and present a set of contrastable observations to address the theory that sequence diversity does lead to functional diversity among 14-3-3 isoforms. The resulting model suggests strongly that there are indeed functional specificities in the 14-3-3s of Arabidopsis. The model further suggests that 14-3-3 diversity and specificity should enter into the discussion of 14-3-3 roles in signal transduction and be directly approached in experiments designed to address 14-3-3 functional roles. It is hoped that future studies involving 14-3-3s will continue to address specificity in experimental design and analysis.

  1. 14-3-3 phosphoprotein interaction networks - does isoform diversity present functional interaction specification?

    Science.gov (United States)

    Paul, Anna-Lisa; Denison, Fiona C; Schultz, Eric R; Zupanska, Agata K; Ferl, Robert J

    2012-01-01

    The 14-3-3 proteins have emerged as major phosphoprotein interaction proteins and thereby constitute a key node in the Arabidopsis Interactome Map, a node through which a large number of important signals pass. Throughout their history of discovery and description, the 14-3-3s have been described as protein families and there has been some evidence that the different 14-3-3 family members within any organism might carry isoform-specific functions. However, there has also been evidence for redundancy of 14-3-3 function, suggesting that the perceived 14-3-3 diversity may be the accumulation of neutral mutations over evolutionary time and as some 14-3-3 genes develop tissue or organ-specific expression. This situation has led to a currently unresolved question - does 14-3-3 isoform sequence diversity indicate functional diversity at the biochemical or cellular level? We discuss here some of the key observations on both sides of the resulting debate, and present a set of contrastable observations to address the theory functional diversity does exist among 14-3-3 isoforms. The resulting model suggests strongly that there are indeed functional specificities in the 14-3-3s of Arabidopsis. The model further suggests that 14-3-3 diversity and specificity should enter into the discussion of 14-3-3 roles in signal transduction and be directly approached in 14-3-3 experimentation. It is hoped that future studies involving 14-3-3s will continue to address specificity in experimental design and analysis. PMID:22934100

  2. Characterization of Affinity-Purified Isoforms of Acinetobacter calcoaceticus Y1 Glutathione Transferases

    OpenAIRE

    Chin-Soon Chee; Irene Kit-Ping Tan; Zazali Alias

    2014-01-01

    Glutathione transferases (GST) were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was r...

  3. Branchial Expression Patterns of Claudin Isoforms in Atlantic Salmon During Seawater Acclimation and Smoltification

    DEFF Research Database (Denmark)

    Tipsmark, Christian K; Kiilerich, Pia; Nilsen, Tom O;

    2008-01-01

    in epithelia. We identified Atlantic salmon genes belonging to the claudin family by screening expressed sequence tag libraries available at NCBI and classification was performed with aid of maximum likelihood and neighbour-joining analysis. In gill libraries, five isoforms (10e, 27a, 28a, 28b and 30) were...... is an important component of cation selective channels while reduction in claudin 27a and 30 may change permeability conditions in favour of the ion secretory mode of the SW gill. Key words: osmoregulation, teleost, tight junction, epithelia, QPCR....

  4. Comparison of fecal pyruvate kinase isoform M2 and calprotectin in acute diarrhea in hospitalized children

    OpenAIRE

    Czub, Elzbieta; Jan K. Nowak; Moczko, Jerzy; Lisowska, Aleksandra; Banaszkiewicz, Aleksandra; Banasiewicz, Tomasz; Walkowiak, Jaroslaw

    2014-01-01

    Fecal concentrations of pyruvate kinase isoform M2 (M2-PK) and calprotectin (FC) serve as biomarkers of inflammation of gastrointestinal mucosa. The value of M2-PK in discriminating between patients with viral and bacterial acute diarrhea (AD) is currently unknown. We analyzed M2-PK and FC concentrations in fifty hospitalized children with AD (29 of which were caused by rotavirus and 21 by Salmonella enteritidis) as well as 32 healthy subjects. There was no difference in the areas under the r...

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

    DEFF Research Database (Denmark)

    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...... siRNA encapsulated in lipid nanoparticles was sufficient to blunt the growth human HCC in a murine xenograft model. Our findings offer preclinical proof-of-concept for HDAC2 blockade as a systemic therapy for liver cancer....

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

    OpenAIRE

    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 ...

  7. Distribution of myosin heavy chain isoforms in muscular dystrophy: insights into disease pathology

    Science.gov (United States)

    Beedle, Aaron M

    2016-01-01

    Myosin heavy chain isoforms are an important component defining fiber type specific properties in skeletal muscle, such as oxidative versus glycolytic metabolism, rate of contraction, and fatigability. While the molecular mechanisms that underlie specification of the different fiber types are becoming clearer, how this programming becomes disrupted in muscular dystrophy and the functional consequences of fiber type changes in disease are not fully resolved. Fiber type changes in disease, with specific focus on muscular dystrophies caused by defects in the dystrophin glycoprotein complex, are discussed. PMID:27430020

  8. Identification of p53 and Its Isoforms in Human Breast Carcinoma Cells

    OpenAIRE

    Zorka Milićević; Vladan Bajić; Lada Živković; Jelena Kasapović; Uroš Andjelković; Biljana Spremo-Potparević

    2014-01-01

    In breast carcinoma, disruption of the p53 pathway is one of the most common genetic alterations. The observation that the p53 can express multiple protein isoforms adds a novel level of complexity to the outcome of p53 mutations. p53 expression was analysed by Western immunoblotting and immunohistochemistry using monoclonal antibodies DO-7, Pab240, and polyclonal antiserum CM-1. The more frequently p53-positive nuclear staining has been found in the invasive breast tumors. One of the most in...

  9. Isoforms and Functions of NAD(P)H Oxidase at the Macula Densa

    OpenAIRE

    Zhang, Rui; Harding, Pamela; Garvin, Jeffery L.; Juncos, Ramiro; Peterson, Ed; Juncos, Luis A.; Liu, Ruisheng

    2009-01-01

    Macula densa cells produce superoxide (O2-) during tubuloglomerular feedback primarily via NAD(P)H oxidase (NOX). The purpose of the present study was to determine NOXs expressed by the macula densa, and the role of each one in NaCl-induced O2- production. To identify which isoforms are expressed, we applied single cell RT-PCR to macula densa cells isolated by laser capture microdissection, and to MMDD1 cells (a macula densa-like cell line). The captured cells expressed nNOS (marker of macula...

  10. PI3K in cancer: divergent roles of isoforms, modes of activation, and therapeutic targeting

    Science.gov (United States)

    Thorpe, Lauren M.; Yuzugullu, Haluk; Zhao, Jean J.

    2015-01-01

    Preface Phosphatidylinositol 3-Kinases (PI3Ks) are critical coordinators of intracellular signaling in response to extracellular stimuli. Hyperactivation of PI3K signaling cascades is one of the most common events in human cancers. In this Review, we discuss recent advances in our knowledge of the roles of distinct PI3K isoforms in normal and oncogenic signaling, the different ways in which PI3K can be upregulated, and the current state and future potential of targeting this pathway in the clinic. PMID:25533673

  11. ApoE Isoform-Dependent Deficits in Extinction of Contextual Fear Conditioning

    OpenAIRE

    Olsen, Reid H. J.; Agam, Mati; Davis, Matthew James; Raber, Jacob

    2012-01-01

    The three major human apoE isoforms (apoE2, apoE3, and apoE4) are encoded by distinct alleles (ε2, ε3, and ε4). Compared to ε3, ε4 is associated with increased risk to develop Alzheimer’s disease (AD), cognitive impairments in Parkinson’s disease (PD), and other conditions. In contrast, a recent study indicated an increased susceptibility to the recurring and re-experiencing symptom cluster of Post Traumatic Stress Disorder (PTSD), as well as related memory impairments, in patients carrying a...

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  14. Transcriptional profiles of glutathione-S-Transferase isoforms, Cyp, and AOE genes in atrazine-exposed zebrafish embryos.

    Science.gov (United States)

    Glisic, Branka; Hrubik, Jelena; Fa, Svetlana; Dopudj, Nela; Kovacevic, Radmila; Andric, Nebojsa

    2016-02-01

    Glutathione-S-transferase (GST) superfamily consists of multiple members involved in xenobiotic metabolism. Expressional pattern of the GST isoforms in adult fish has been used as a biomarker of exposure to environmental chemicals. However, GST transcriptional responses vary across organs, thus requiring a cross-tissue examination of multiple mRNAs for GST profiling in an animal after chemical exposure. Zebrafish embryos express all GST isoforms as adult fish and could therefore represent an alternative model for identification of biomarkers of exposure. To evaluate such a possibility, we studied a set of cytosolic and microsomal GST isoform-specific expression profiles in the zebrafish embryos after exposure to atrazine, a widely used herbicide. Expression of the GST isoforms was compared with that of CYP genes involved in the phase I of xenobiotic metabolism and antioxidant enzyme (AOE) genes. Using quantitative real-time PCR, we showed dynamic changes in the expressional pattern of twenty GST isoforms, cyp1a, cyp3a65, ahr2, and four AOEs in early development of zebrafish. Acute (48 and 72 h) exposure of 24 h-old embryos to atrazine, from environmentally relevant (0.005 mg/L) to high (40 mg/L) concentrations, caused a variety of transient, albeit minor changes (atrazine (5 and 40 mg/L). In summary, an analysis of the response of multiple systems in the zebrafish embryos provided a comprehensive understanding of atrazine toxicity and its potential impact on biological processes.

  15. Human-specific protein isoforms produced by novel splice sites in the human genome after the human-chimpanzee divergence

    Directory of Open Access Journals (Sweden)

    Kim Dong Seon

    2012-11-01

    Full Text Available Abstract Background Evolution of splice sites is a well-known phenomenon that results in transcript diversity during human evolution. Many novel splice sites are derived from repetitive elements and may not contribute to protein products. Here, we analyzed annotated human protein-coding exons and identified human-specific splice sites that arose after the human-chimpanzee divergence. Results We analyzed multiple alignments of the annotated human protein-coding exons and their respective orthologous mammalian genome sequences to identify 85 novel splice sites (50 splice acceptors and 35 donors in the human genome. The novel protein-coding exons, which are expressed either constitutively or alternatively, produce novel protein isoforms by insertion, deletion, or frameshift. We found three cases in which the human-specific isoform conferred novel molecular function in the human cells: the human-specific IMUP protein isoform induces apoptosis of the trophoblast and is implicated in pre-eclampsia; the intronization of a part of SMOX gene exon produces inactive spermine oxidase; the human-specific NUB1 isoform shows reduced interaction with ubiquitin-like proteins, possibly affecting ubiquitin pathways. Conclusions Although the generation of novel protein isoforms does not equate to adaptive evolution, we propose that these cases are useful candidates for a molecular functional study to identify proteomic changes that might bring about novel phenotypes during human evolution.

  16. Two isoforms of TALDO1 generated by alternative translational initiation show differential nucleocytoplasmic distribution to regulate the global metabolic network

    Science.gov (United States)

    Moriyama, Tetsuji; Tanaka, Shu; Nakayama, Yasumune; Fukumoto, Masahiro; Tsujimura, Kenji; Yamada, Kohji; Bamba, Takeshi; Yoneda, Yoshihiro; Fukusaki, Eiichiro; Oka, Masahiro

    2016-01-01

    Transaldolase 1 (TALDO1) is a rate-limiting enzyme involved in the pentose phosphate pathway, which is traditionally thought to occur in the cytoplasm. In this study, we found that the gene TALDO1 has two translational initiation sites, generating two isoforms that differ by the presence of the first 10 N-terminal amino acids. Notably, the long and short isoforms were differentially localised to the cell nucleus and cytoplasm, respectively. Pull-down and in vitro transport assays showed that the long isoform, unlike the short one, binds to importin α and is actively transported into the nucleus in an importin α/β-dependent manner, demonstrating that the 10 N-terminal amino acids are essential for its nuclear localisation. Additionally, we found that these two isoforms can form homo- and/or hetero-dimers with different localisation dynamics. A metabolite analysis revealed that the subcellular localisation of TALDO1 is not crucial for its activity in the pentose phosphate pathway. However, the expression of these two isoforms differentially affected the levels of various metabolites, including components of the tricarboxylic acid cycle, nucleotides, and sugars. These results demonstrate that the nucleocytoplasmic distribution of TALDO1, modulated via alternative translational initiation and dimer formation, plays an important role in a wide range of metabolic networks. PMID:27703206

  17. The expression of ELK transcription factors in adult DRG: Novel isoforms, antisense transcripts and upregulation by nerve damage.

    Science.gov (United States)

    Kerr, Niall; Pintzas, Alexander; Holmes, Fiona; Hobson, Sally-Ann; Pope, Robert; Wallace, Mark; Wasylyk, Christine; Wasylyk, Bohdan; Wynick, David

    2010-06-01

    ELK transcription factors are known to be expressed in a number of regions in the nervous system. We show by RT-PCR that the previously described Elk1, Elk3/Elk3b/Elk3c and Elk4 mRNAs are expressed in adult dorsal root ganglia (DRG), together with the novel alternatively spliced isoforms Elk1b, Elk3d and Elk4c/Elk4d/Elk4e. These isoforms are also expressed in brain, heart, kidney and testis. In contrast to Elk3 protein, the novel Elk3d isoform is cytoplasmic, fails to bind ETS binding sites and yet can activate transcription by an indirect mechanism. The Elk3 and Elk4 genes are overlapped by co-expressed Pctk2 (Cdk17) and Mfsd4 genes, respectively, with the potential formation of Elk3/Pctaire2 and Elk4/Mfsd4 sense-antisense mRNA heteroduplexes. After peripheral nerve injury the Elk3 mRNA isoforms are each upregulated approximately 2.3-fold in DRG (P<0.005), whereas the natural antisense Pctaire2 isoforms show only a small increase (21%, P<0.01) and Elk1 and Elk4 mRNAs are unchanged. PMID:20304071

  18. Alternative mRNA Splicing from the Glial Fibrillary Acidic Protein (GFAP) Gene Generates Isoforms with Distinct Subcellular mRNA Localization Patterns in Astrocytes

    DEFF Research Database (Denmark)

    Thomsen, Rune; Daugaard, Tina Fuglsang; Holm, Ida E;

    2013-01-01

    The intermediate filament network of astrocytes includes Glial fibrillary acidic protein (Gfap) as a major component. Gfap mRNA is alternatively spliced resulting in generation of different protein isoforms where Gfapa is the most predominant isoform. The Gfapd isoform is expressed in proliferating......RNA localization patterns were dependent on the different 39-exon sequences included in Gfapd and Gfapa mRNA. The presented results show that alternative Gfap mRNA splicing results in isoform-specific mRNA localization patterns with resulting different local mRNA concentration ratios which have potential...

  19. A novel isoform of MAP4 organises the paraxial microtubule array required for muscle cell differentiation.

    Science.gov (United States)

    Mogessie, Binyam; Roth, Daniel; Rahil, Zainab; Straube, Anne

    2015-01-01

    The microtubule cytoskeleton is critical for muscle cell differentiation and undergoes reorganisation into an array of paraxial microtubules, which serves as template for contractile sarcomere formation. In this study, we identify a previously uncharacterised isoform of microtubule-associated protein MAP4, oMAP4, as a microtubule organising factor that is crucial for myogenesis. We show that oMAP4 is expressed upon muscle cell differentiation and is the only MAP4 isoform essential for normal progression of the myogenic differentiation programme. Depletion of oMAP4 impairs cell elongation and cell-cell fusion. Most notably, oMAP4 is required for paraxial microtubule organisation in muscle cells and prevents dynein- and kinesin-driven microtubule-microtubule sliding. Purified oMAP4 aligns dynamic microtubules into antiparallel bundles that withstand motor forces in vitro. We propose a model in which the cooperation of dynein-mediated microtubule transport and oMAP4-mediated zippering of microtubules drives formation of a paraxial microtubule array that provides critical support for the polarisation and elongation of myotubes. PMID:25898002

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

    Science.gov (United States)

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

    2015-01-01

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

  1. Secondary structural analysis of the carboxyl-terminal domain from different connexin isoforms.

    Science.gov (United States)

    Spagnol, Gaëlle; Al-Mugotir, Mona; Kopanic, Jennifer L; Zach, Sydney; Li, Hanjun; Trease, Andrew J; Stauch, Kelly L; Grosely, Rosslyn; Cervantes, Matthew; Sorgen, Paul L

    2016-03-01

    The connexin carboxyl-terminal (CxCT) domain plays a role in the trafficking, localization, and turnover of gap junction channels, as well as the level of gap junction intercellular communication via numerous post-translational modifications and protein-protein interactions. As a key player in the regulation of gap junctions, the CT presents itself as a target for manipulation intended to modify function. Specific to intrinsically disordered proteins, identifying residues whose secondary structure can be manipulated will be critical toward unlocking the therapeutic potential of the CxCT domain. To accomplish this goal, we used biophysical methods to characterize CxCT domains attached to their fourth transmembrane domain (TM4). Circular dichroism and nuclear magnetic resonance were complementary in demonstrating the connexin isoforms that form the greatest amount of α-helical structure in their CT domain (Cx45 > Cx43 > Cx32 > Cx50 > Cx37 ≈ Cx40 ≈ Cx26). Studies compared the influence of 2,2,2-trifluoroethanol, pH, phosphorylation, and mutations (Cx32, X-linked Charcot-Marie Tooth disease; Cx26, hearing loss) on the TM4-CxCT structure. While pH modestly influences the CT structure, a major structural change was associated with phosphomimetic substitutions. Since most connexin CT domains are phosphorylated throughout their life cycle, studies of phospho-TM4-CxCT isoforms will be critical toward understanding the role that structure plays in regulating gap junction function. PMID:26542351

  2. Inhibition of Ptychopetalum olacoides on Acetylcholinesterase Isoforms in Brain of Mice

    Institute of Scientific and Technical Information of China (English)

    FIGUEIR(O) Micheli; POCHMANN Daniela; PORCI(U)NCULA Lisiane Oliveira; NUNES Domingos Sávio; ELISABESTKY Elaine

    2012-01-01

    Objective To further characterize the acetylcholinesterase inhibitors (AChE-Is) pattern of Ptychopetalum olacoides ethanol extract (POEE) on the cytosolic globular monomer (G1) and membrane bound globular tetramer (G4) AChE isoforms in brain areas relevant for cognition.Methods The G1and G4 AChE isoforms were prepared according to the reported methods and the determination of AChE activity used was adapted from colorimetric method.Results POEE mostly inhibited G1in hippocampus (75%),and G4 in frontal cortex (58%) and striatum (75%) (P < 0.05).Kinetic analysis indicated that POEE-induced AChE inhibition in hippocampus was of a competitive nature for G1but uncompetitive for G4.Conclusion Considering the high density of cholinergic projection to the cortex and striatum,and the usefulness of conserving cytosolic acetylcholine to replenish synaptic vesicles in a highly active cognition site such as hippocampus,we argue that this could be a desirable profile for a clinically relevant AChE-I.

  3. Mitochondrial Sulfide Detoxification Requires a Functional Isoform O-Acetylserine(thiol)lyase C in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Consolación (A)lvarez; Irene García; Luis C.Romero; Cecilia Gotor

    2012-01-01

    In non-cyanogenic species,the main source of cyanide derives from ethylene and camalexin biosyntheses.In mitochondria,cyanide is a potent inhibitor of the cytochrome c oxidase and is metabolized bythe β-cyanoalanine synthase CYS-C1,catalyzing the conversion of cysteine and cyanide to hydrogen sulfide and β-cyanoalanine.The hydrogen sulfide released also inhibits the cytochrome c oxidase and needs to be detoxified by the O-acetylserine(thiol)lyase mitochondrial isoform,OAS-C,which catalyzes the incorporation of sulfide to O-acetylserine to produce cysteine,thus generating a cyclic pathway in the mitochondria.The loss of functional OAS-C isoforms causes phenotypic characteristics very similar to the loss of the CYS-C1 enzyme,showing defects in root hair formation.Genetic complementation with the OAS-C gene rescues the impairment of root hair elongation,restoring the wild-type phenotype.The mitochondria compromise their capacity to properly detoxify cyanide and the resulting sulfide because the latter cannot re-assimilate into cysteine in the oas-c null mutant.Consequently,we observe an accumulation of sulfide and cyanide and of the alternative oxidase,which is unable to prevent the production of reactive oxygen species probably due to the accumulation of both toxic molecules.Our results allow us to suggest that the significance of OAS-C is related to its role in the proper sulfide and cyanide detoxification in mitochondria.

  4. Splice isoforms of the polyglutamine disease protein ataxin-3 exhibit similar enzymatic yet different aggregation properties.

    Directory of Open Access Journals (Sweden)

    Ginny Marie Harris

    Full Text Available Protein context clearly influences neurotoxicity in polyglutamine diseases, but the contribution of alternative splicing to this phenomenon has rarely been investigated. Ataxin-3, a deubiquitinating enzyme and the disease protein in SCA3, is alternatively spliced to encode either a C-terminal hydrophobic stretch or a third ubiquitin interacting motif (termed 2UIM and 3UIM isoforms, respectively. In light of emerging insights into ataxin-3 function, we examined the significance of this splice variation. We confirmed neural expression of several minor 5' variants and both of the known 3' ataxin-3 splice variants. Regardless of polyglutamine expansion, 3UIM ataxin-3 is the predominant isoform in brain. Although 2UIM and 3UIM ataxin-3 display similar in vitro deubiquitinating activity, 2UIM ataxin-3 is more prone to aggregate and more rapidly degraded by the proteasome. Our data demonstrate how alternative splicing of sequences distinct from the trinucleotide repeat can alter properties of the encoded polyglutamine disease protein and thereby perhaps contribute to selective neurotoxicity.

  5. Somatodendritic and excitatory postsynaptic distribution of neuron-type dystrophin isoform, Dp40, in hippocampal neurons.

    Science.gov (United States)

    Fujimoto, Takahiro; Itoh, Kyoko; Yaoi, Takeshi; Fushiki, Shinji

    2014-09-12

    The Duchenne muscular dystrophy (DMD) gene produces multiple dystrophin (Dp) products due to the presence of several promoters. We previously reported the existence of a novel short isoform of Dp, Dp40, in adult mouse brain. However, the exact biochemical expression profile and cytological distribution of the Dp40 protein remain unknown. In this study, we generated a polyclonal antibody against the NH2-terminal region of the Dp40 and identified the expression profile of Dp40 in the mouse brain. Through an analysis using embryonic and postnatal mouse cerebrums, we found that Dp40 emerged from the early neonatal stages until adulthood, whereas Dp71, an another Dp short isoform, was highly detected in both prenatal and postnatal cerebrums. Intriguingly, relative expressions of Dp40 and Dp71 were prominent in cultured dissociated neurons and non-neuronal cells derived from mouse hippocampus, respectively. Furthermore, the immunocytological distribution of Dp40 was analyzed in dissociated cultured neurons, revealing that Dp40 is detected in the soma and its dendrites, but not in the axon. It is worthy to note that Dp40 is localized along the subplasmalemmal region of the dendritic shafts, as well as at excitatory postsynaptic sites. Thus, Dp40 was identified as a neuron-type Dp possibly involving dendritic and synaptic functions.

  6. Smooth muscle actin isoforms: a tug of war between contraction and compliance.

    Science.gov (United States)

    Arnoldi, Richard; Hiltbrunner, Anita; Dugina, Vera; Tille, Jean-Christophe; Chaponnier, Christine

    2013-01-01

    In higher vertebrates, smooth muscle (SM) contains two tissue-specific actin isoforms: α-SMA and γ-SMA, which predominate in vascular and visceral SM, respectively. Whether α-SMA has been extensively studied and recognized for its contractile activity in SM and SM-like cells such as myofibroblasts, myoepithelial and myoid cells, the distribution and role of γ-SMA remained largely unknown. We developed a new specific monoclonal antibody against γ-SMA and confirmed that γ-SMA predominates in the visceral system and is minor in the vascular system, although more expressed in highly compliant veins than in stiff arteries. Contrary to α-SMA, γ-SMA is absent from myofibroblasts in vitro, and in fibrotic diseases in vivo. We raised the hypothesis that, whereas α-SMA is responsible for the "contractile" activity, γ-SMA would be involved in the "compliance" of SM and SM-like cells. Several models support this hypothesis, namely veins vs. arteries and the physiological modifications occurring in the uterus and mammary glands during pregnancy and lactation. Our results suggest that, in addition to enteric smooth muscles, γ-SMA is expressed in all the tissues submitted to an important dilation including veins, gravid uterus, and lactating mammary glands. The hypothesis of two complementary mechanical roles for the two SMA isoforms is sustained by their different intracellular distributions and by functional assays. PMID:23915964

  7. Subcellular distribution of nitric oxide synthase isoforms in the rat duodenum

    Institute of Scientific and Technical Information of China (English)

    Petra Talapka; Nikolett Bódi; Izabella Battonyai; (E)va Fekete; Mária Bagyánszki

    2011-01-01

    AIM:To study the cell-type specific subcellular distribution of the three isoforms of nitric oxide synthase (NOS) in the rat duodenum. METHODS:Postembedding immunoelectronmicroscopy was performed,in which primary antibodies for neuronal NOS (nNOS),endothelial NOS (eNOS),and inducible NOS (iNOS),were visualized with protein A-gold-conjugated secondary antibodies.Stained ultrathin sections were examined and photographed with a Philips CM10 electron microscope equipped with a MEGAVIEW II camera.The specificity of the immunoreaction in all cases was assessed by omitting the primary antibodies in the labeling protocol and incubating the sections only in the protein A-gold conjugated secondary antibodies. RESULTS:Postembedding immunoelectronmicroscopy revealed the presence of nNOS,eNOS,and iNOS immunoreactivity in the myenteric neurons,the enteric smooth muscle cells,and the endothelium of capillaries running in the vicinity of the myenteric plexus of the rat duodenum.The cell type-specific distributions of the immunogold particles labeling the three different NOS isozymes were revealed.In the control experiments,in which the primary antiserum was omitted,virtually no postembedding gold particles were observed. CONCLUSION:This postembedding immunoelectronmicroscopic study provided the first evidence of celltype- specific differences in the subcellular distributions of NOS isoforms.

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

    Energy Technology Data Exchange (ETDEWEB)

    Bellamine, A.; Lepesheva, Galina I.; Waterman, Mike (Vanderbilt)

    2010-11-16

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

  9. The role of telomeres and telomerase reverse transcriptase isoforms in pluripotency induction and maintenance.

    Science.gov (United States)

    Teichroeb, Jonathan H; Kim, Joohwan; Betts, Dean H

    2016-08-01

    Telomeres are linear guanine-rich DNA structures at the ends of chromosomes. The length of telomeric DNA is actively regulated by a number of mechanisms in highly proliferative cells such as germ cells, cancer cells, and pluripotent stem cells. Telomeric DNA is synthesized by way of the ribonucleoprotein called telomerase containing a reverse transcriptase (TERT) subunit and RNA component (TERC). TERT is highly conserved across species and ubiquitously present in their respective pluripotent cells. Recent studies have uncovered intricate associations between telomeres and the self-renewal and differentiation properties of pluripotent stem cells. Interestingly, the past decade's work indicates that the TERT subunit also has the capacity to modulate mitochondrial function, to remodel chromatin structure, and to participate in key signaling pathways such as the Wnt/β-catenin pathway. Many of these non-canonical functions do not require TERT's catalytic activity, which hints at possible functions for the extensive number of alternatively spliced TERT isoforms that are highly expressed in pluripotent stem cells. In this review, some of the established and potential routes of pluripotency induction and maintenance are highlighted from the perspectives of telomere maintenance, known TERT isoform functions and their complex regulation. PMID:26786236

  10. Analysis of chloroquine resistance transporter (CRT) isoforms and orthologues in S. cerevisiae yeast.

    Science.gov (United States)

    Baro, Nicholas K; Pooput, Chaya; Roepe, Paul D

    2011-08-01

    Previous work from our laboratory optimized MeOH-inducible expression of the P. falciparum malarial parasite transporter PfCRT in P. pastoris yeast. These strains are useful for many experiments but do not allow for inducible protein expression under ambient growth conditions. We have therefore optimized galactose-inducible expression of PfCRT in S. cerevisiae yeast. We find that expression of PfCRT confers CQ hypersensitivity to growing yeast and that this is due to plasma membrane localization of the transporter. We use quantitative analyses of growth rates to compare hypersensitivity for yeast expressing various PfCRT isoforms. We also report successful high level inducible expression of the P. vivax orthologue, PvCRT, and compare CQ hypersensitivity for PvCRT vs PfCRT expressing yeast. We test the hypothesis that hypersensitivity is due to increased transport of CQ into yeast expressing the transporters via direct (3)H-CQ transport experiments and analyze the effect that membrane potential has on transport. The data suggest important new tools for rapid functional screening of PfCRT and PvCRT isoforms and provide further evidence for a model wherein membrane potential promotes charged CQ transport by PfCRT. Data also support our previous conclusion that wild type PfCRT is capable of CQ transport and provide a basis for understanding the lack of correspondence between PvCRT mutations and resistance to CQ in the important malarial parasite P. vivax.

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

    Science.gov (United States)

    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.

  12. Data on CUX1 isoforms in idiopathic pulmonary fibrosis lung and systemic sclerosis skin tissue sections.

    Science.gov (United States)

    Ikeda, Tetsurou; Fragiadaki, Maria; Shi-Wen, Xu; Ponticos, Markella; Khan, Korsa; Denton, Christopher; Garcia, Patricia; Bou-Gharios, George; Yamakawa, Akio; Morimoto, Chikao; Abraham, David

    2016-09-01

    This data article contains complementary figures related to the research article entitled, "Transforming growth factor-β-induced CUX1 isoforms are associated with fibrosis in systemic sclerosis lung fibroblasts" (Ikeda et al. (2016) [2], http://dx.doi.org/10.1016/j.bbrep.2016.06.022), which presents that TGF-β increased CUX1 binding in the proximal promoter and enhancer of the COL1A2 and regulated COL1. Further, in the scleroderma (SSc) lung and diffuse alveolar damage lung sections, CUX1 localized within the α- smooth muscle actin (α-SMA) positive cells (Fragiadaki et al., 2011) [1], "High doses of TGF-beta potently suppress type I collagen via the transcription factor CUX1" (Ikeda et al., 2016) [2]. Here we show that CUX1 isoforms are localized within α-smooth muscle actin-positive cells in SSc skin and idiopathic pulmonary fibrosis (IPF) lung tissue sections. In particular, at the granular and prickle cell layers in the SSc skin sections, CUX1 and α-SMA are co-localized. In addition, at the fibrotic loci in the IPF lung tissue sections, CUX1 localized within the α-smooth muscle actin (α-SMA) positive cells. PMID:27583344

  13. Myophilin of Echinococcus granulosus: isoforms and phosphorylation by protein kinase C.

    Science.gov (United States)

    Martin, R M; Csar, X F; Gasser, R B; Felleisen, R; Lightowlers, M W

    1997-08-01

    Myophilin is a muscle-associated antigen of the taeniid cestode Echinococcus granulosus. This protein shows a high amino acid sequence homology with calponins and calponin-like proteins, which are proposed to be associated with the regulation of smooth muscle contraction. In order to provide supportive evidence for a relationship between these proteins, we characterized myophilin using electrophoretic, biochemical and molecular biological approaches. Two-dimensional protein electrophoretic separation of E. granulosus larval proteins defined 4 isoelectric isoforms of myophilin (alpha, beta, gamma and delta), which appeared to be a consequence of post-translational modification of a single gene product. It was also demonstrated biochemically that E. granulosus myophilin undergoes specific phosphorylation in vitro by protein kinase C (PKC). Finally, myophilin homologues were identified in extracts of Taenia hydatigena and T. ovis by immunoblot. A partial cDNA of the closely related species, E. multilocularis, was isolated by cloning procedures and showed 99% homology with the E. granulosus myophilin gene. The similarities of E. granulosus myophilin with calponins in their tissue localization, protein isoforms patterns, ability to be phosphorylated in vitro by PKC, and the relatively conserved nature of the protein among related parasites suggest that myophilin may be associated with smooth muscle contraction.

  14. High-throughput proteomics detection of novel splice isoforms in human platelets.

    LENUS (Irish Health Repository)

    Power, Karen A

    2009-01-01

    Alternative splicing (AS) is an intrinsic regulatory mechanism of all metazoans. Recent findings suggest that 100% of multiexonic human genes give rise to splice isoforms. AS can be specific to tissue type, environment or developmentally regulated. Splice variants have also been implicated in various diseases including cancer. Detection of these variants will enhance our understanding of the complexity of the human genome and provide disease-specific and prognostic biomarkers. We adopted a proteomics approach to identify exon skip events - the most common form of AS. We constructed a database harboring the peptide sequences derived from all hypothetical exon skip junctions in the human genome. Searching tandem mass spectrometry (MS\\/MS) data against the database allows the detection of exon skip events, directly at the protein level. Here we describe the application of this approach to human platelets, including the mRNA-based verification of novel splice isoforms of ITGA2, NPEPPS and FH. This methodology is applicable to all new or existing MS\\/MS datasets.

  15. HLA-G5 and G7 Isoforms in Pregnant Women.

    Directory of Open Access Journals (Sweden)

    Saeid Abediankenari

    2015-04-01

    Full Text Available Human leukocyte antigen-G which is an immune tolerance effecter molecule has an important role in the maintenance of fetus during pregnancy. Abortion is one of the complications of pregnancy period.  In this research, we have studied levels of HLA-G 5, HLA-G7 isoforms in the abortion-threatened pregnant women in comparison with controls. In a case-control study, 101 abortion-threatened women and 101 healthy pregnant women (healthy controls with age range 21-32 years were studied. Gene expression of HLA-5 and HLA-7 isoforms was analyzed by real-time polymerase chain reaction after mRNA extraction and cDNA synthesis. The results indicated that HLA-G5 was significantly lower in abortion-threatened women in comparison with the control group whereas HLA-G7 was not significantly differentbetween the 2 groups. HLA-G is a vital molecule during pregnancy that can be a key factor in prevention of abortion. It is concluded that determination of HLA-G5 can be of value in pregnancy.

  16. Soluble expression and characterization of a GFP-fused pea actin isoform(PEAc1)

    Institute of Scientific and Technical Information of China (English)

    Ai Xiao LIU; Shao Bin ZHANG; Xiao Jing XU; Dong Tao REN; Guo Qin LIU

    2004-01-01

    A pea actin isoform PEAc1 with green fluorescent protein (GFP) fusion to its C-terminus and His-tag to its Nterminus, was expressed in prokaryotic cells in soluble form, and highly purified with Ni-Chelating SepharoseTM Fast Flow column. The purified fusion protein (PEAc1-GFP) efficiently inhibited DNase I activities before polymerization,and activated the myosin Mg-ATPase activities after polymerization. The PEAc 1-GFP also polymerized into green fluorescent filamentous structures with a critical concentration of 0.75 μM. These filamentous structures were labeled by TRITC-phalloidin, a specific agent for staining actin microfilaments, and identified as having 9 nm diameters by negative staining. These results indicated that PEAc1 preserved the essential characteristics of actin even with His-tag and GFP fusion, suggesting a promising potential to use GFP fusion protein in obtainning soluble plant actin isoform to analyze its physical and biochemical properties in vitro. The PEAc 1-GFP was also expressed in tobacco BY2 cells,which offers a new pathway for further studying its distribution and function in vivo.

  17. Interaction of myelin basic protein isoforms with lipid bilayers studied by FTIR spectroscopy

    Science.gov (United States)

    Jackson, Michael; Choo, Lin-P'ing; Boulias, Christopher; Moscarello, Mario A.; Mantsch, Henry H.

    1993-05-01

    The secondary structure of the naturally occurring isoforms of myelin basic protein (MBP1-8) from human myelin was studied by Fourier transform infrared spectroscopy under a variety of experimental conditions. In aqueous solution each isoform was found to be unstructured. In the presence of negatively charged liquid bilayers MBP1-4 were shown to exhibit an amide I band maximum indicative of the adoption of (alpha) -helical secondary structures. A detailed analysis revealed that significant proportions of (beta) -sheet secondary structure were also present. MBP5 and MBP8, which have significantly less cationic charge than MBP1-4, exhibited an amide I maximum identical to that seen in solution, suggesting that no interaction with the bilayer occurred. Analysis of the lipid CH2 and C equals O stretching vibrations also pointed towards significant interaction of MBP1-4 with the bilayer. The changes in intensity and frequency of these bands which typically accompany the phase transition in the pure bilayer were abolished by addition of the proteins. No such effect was seen for MBP5 and 8, the normal lipid phase transition being apparent. The implications of these results in the aetiology of multiple sclerosis is discussed.

  18. Distinct Functions of Different scl Isoforms in Zebrafish Definitive Hematopoietic Stem Cell Initiation and Maintenance

    Science.gov (United States)

    Lan, Yahui

    2011-07-01

    The establishment of entire blood system relies on the multi-potent hematopoietic stem cells (HSCs), thus identifying the molecular mechanism in HSC generation is of importance for not only complementing the fundamental knowledge in stem cell biology, but also providing insights to the regenerative therapies. Recent researches have documented the formation of nascent HSCs through a direct transition from ventral aortic endothelium, named as endothelial hematopoietic transition (EHT) process. However, the precise genetic program engaged in this process remains largely elusive. The transcription factor scl plays pivotal and conserved roles in embryonic and adult hematopoiesis from teleosts to mammals. Our lab have previously identified a new truncated scl isoform, scl-beta, which is indispensible for the specification of HSCs in the ventral wall of dorsal aorta (VDA), the zebrafish equivalent of mammalian fetal hematopoietic organ. Here we observe that, by combining time-lapse confocal imaging of transgenic zebrafish and genetic epistasis analysis, scl-beta is expressed in a subset of ventral aortic endothelial cells and critical for their forthcoming transformation to hemogenic endothelium; in contrast, runx1 is required downstream to govern the successful egress of the hemogenic endothelial cells to become naive HSCs. In addition, the traditional known full-length scl-alpha isoform is firstly evidenced to be required for the maintenance or survival of newly formed HSCs in VDA. Collectively our data has established the genetic hierarchy controlling discrete steps in the consecutive process of HSC formation from endothelial cells and further development in VDA.

  19. Somatodendritic and excitatory postsynaptic distribution of neuron-type dystrophin isoform, Dp40, in hippocampal neurons.

    Science.gov (United States)

    Fujimoto, Takahiro; Itoh, Kyoko; Yaoi, Takeshi; Fushiki, Shinji

    2014-09-12

    The Duchenne muscular dystrophy (DMD) gene produces multiple dystrophin (Dp) products due to the presence of several promoters. We previously reported the existence of a novel short isoform of Dp, Dp40, in adult mouse brain. However, the exact biochemical expression profile and cytological distribution of the Dp40 protein remain unknown. In this study, we generated a polyclonal antibody against the NH2-terminal region of the Dp40 and identified the expression profile of Dp40 in the mouse brain. Through an analysis using embryonic and postnatal mouse cerebrums, we found that Dp40 emerged from the early neonatal stages until adulthood, whereas Dp71, an another Dp short isoform, was highly detected in both prenatal and postnatal cerebrums. Intriguingly, relative expressions of Dp40 and Dp71 were prominent in cultured dissociated neurons and non-neuronal cells derived from mouse hippocampus, respectively. Furthermore, the immunocytological distribution of Dp40 was analyzed in dissociated cultured neurons, revealing that Dp40 is detected in the soma and its dendrites, but not in the axon. It is worthy to note that Dp40 is localized along the subplasmalemmal region of the dendritic shafts, as well as at excitatory postsynaptic sites. Thus, Dp40 was identified as a neuron-type Dp possibly involving dendritic and synaptic functions. PMID:25152393

  20. The combined expression patterns of Ikaros isoforms characterize different hematological tumor subtypes.

    Directory of Open Access Journals (Sweden)

    Carlos A Orozco

    Full Text Available A variety of genetic alterations are considered hallmarks of cancer development and progression. The Ikaros gene family, encoding for key transcription factors in hematopoietic development, provides several examples as genetic defects in these genes are associated with the development of different types of leukemia. However, the complex patterns of expression of isoforms in Ikaros family genes has prevented their use as clinical markers. In this study, we propose the use of the expression profiles of the Ikaros isoforms to classify various hematological tumor diseases. We have standardized a quantitative PCR protocol to estimate the expression levels of the Ikaros gene exons. Our analysis reveals that these levels are associated with specific types of leukemia and we have found differences in the levels of expression relative to five interexonic Ikaros regions for all diseases studied. In conclusion, our method has allowed us to precisely discriminate between B-ALL, CLL and MM cases. Differences between the groups of lymphoid and myeloid pathologies were also identified in the same way.

  1. Isolation and characterization of cytosolic alanine aminotransferase isoforms from starved rat liver.

    Science.gov (United States)

    Vedavathi, M; Girish, K S; Kumar, M Karuna

    2004-12-01

    Alanine is the most effective precursor for gluconeogenesis among amino acids and the initial reaction is catalyzed by alanine aminotransferases (AlaATs). It is a less extensively studied enzyme under starvation and known to that the enzyme activity increases in liver under starvation. The present study describes the purification and characterization of two isoforms of alanine aminotransferases from starved male rat liver under starvation. The molecular mass of isoforms was found to be 17.7 and 112.2 kDa with isoelectric points of 4.2 and 5.3 respectively for AlaAT I and AlaAT II. Both the enzymes showed narrow substrate specificity for L-alanine with different Km for alanine and 2-oxoglutarate. Both the enzymes were glycoprotein in nature. Inhibition, modification and spectroscopic studies showed that both PLP and free-SH groups are directly involved in the enzymatic catalysis. PLP activated both the enzymes with a Km 0.057 mM and 0.2 mM for AlaAT I and II respectively. PMID:15663181

  2. Investigating the role of class-IA PI 3-kinase isoforms in adipocyte differentiation

    International Nuclear Information System (INIS)

    PI 3-kinases, in particular class-IA, are key signalling molecules controlling many cellular processes including growth, proliferation, migration and differentiation. In this study, we have used a collection of isoform selective PI 3-kinase inhibitors to determine whether attenuation of signalling through class-IA PI 3-kinase isoforms will impact adipocyte differentiation. First, we analysed the expression profiles and found that fibroblastic pre-adipocytes express detectable levels of p110α and p110δ and that after differentiation, p110δ levels fall while p110α levels rise, together with C/EBPα and PPARγ. When using specific inhibitors during the differentiation process, we observed that neither p110β nor p110δ inhibition, had any significant effect. In contrast PIK-75, a selective p110α inhibitor completely abolished adipocyte differentiation as assessed by morphology, transcript and protein levels of adipocyte markers. These results indicate that long term treatment with p110α inhibitors could potentially have a severe impact on fat cell numbers in vivo.

  3. Expression of VEGF(xxx)b, the inhibitory isoforms of VEGF, in malignant melanoma.

    Science.gov (United States)

    Pritchard-Jones, R O; Dunn, D B A; Qiu, Y; Varey, A H R; Orlando, A; Rigby, H; Harper, S J; Bates, D O

    2007-07-16

    Malignant melanoma is the most lethal of the skin cancers and the UK incidence is rising faster than that of any other cancer. Angiogenesis - the growth of new vessels from preexisting vasculature - is an absolute requirement for tumour survival and progression beyond a few hundred microns in diameter. We previously described a class of anti-angiogenic isoforms of VEGF, VEGF(xxx)b, that inhibit tumour growth in animal models, and are downregulated in some cancers, but have not been investigated in melanoma. To determine whether VEGF(xxx)b expression was altered in melanoma, PCR and immunohistochemistry of archived human tumour samples were used. In normal epidermis and in a proportion of melanoma samples, VEGF(xxx)b staining was seen. Some melanomas had much weaker staining. Subsequent examination revealed that expression was significantly reduced in primary melanoma samples (both horizontal and vertical growth phases) from patients who subsequently developed tumour metastasis compared with those who did not (analysis of variance (ANOVA) Pxxx)b expression appears to predict metastatic spread in patients with primary melanoma. These results suggest that there is a switch in splicing as part of the metastatic process, from anti-angiogenic to pro-angiogenic VEGF isoforms. This may form part of a wider metastatic splicing phenotype.

  4. Effects of contraction on localization of GLUT4 and v-SNARE isoforms in rat skeletal muscle

    DEFF Research Database (Denmark)

    Rose, Adam John; Jeppesen, Jacob; Kiens, Bente;

    2009-01-01

    In skeletal muscle, contractions increase glucose uptake due to a translocation of GLUT4 glucose transporters from intracellular storage sites to the surface membrane. Vesicle associated membrane proteins (VAMPs) are believed to play an important role in docking and fusion of the GLUT4 transporters...... at the surface membrane. However, knowledge about which VAMP isoforms in fact co-localize with GLUT4 vesicles in mature skeletal muscle and whether they translocate during muscle contractions is incomplete. The aim of the present study was to further identify VAMP isoforms which are associated with GLUT4...... vesicles and examine which VAMP isoforms translocate to surface membranes in skeletal muscles undergoing contractions. VAMP2, VAMP3, VAMP5 and VAMP7 were enriched in immuno-precipitated GLUT4 vesicles. In response to 20 min of in situ contractions, there was a redistribution of GLUT4 (+64 +/- 13...

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

    Energy Technology Data Exchange (ETDEWEB)

    Faddy, Helen M.; Smart, Chanel E.; Xu, Ren; Lee, Genee Y.; Kenny, Paraic A.; Feng, Mingye; Rao, Rajini; Brown, Melissa A.; Bissell, Mina J.; Roberts-Thomson, Sarah J.; Monteith, Gregory R.

    2008-04-09

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

  6. Transcriptional profiles of glutathione-S-Transferase isoforms, Cyp, and AOE genes in atrazine-exposed zebrafish embryos.

    Science.gov (United States)

    Glisic, Branka; Hrubik, Jelena; Fa, Svetlana; Dopudj, Nela; Kovacevic, Radmila; Andric, Nebojsa

    2016-02-01

    Glutathione-S-transferase (GST) superfamily consists of multiple members involved in xenobiotic metabolism. Expressional pattern of the GST isoforms in adult fish has been used as a biomarker of exposure to environmental chemicals. However, GST transcriptional responses vary across organs, thus requiring a cross-tissue examination of multiple mRNAs for GST profiling in an animal after chemical exposure. Zebrafish embryos express all GST isoforms as adult fish and could therefore represent an alternative model for identification of biomarkers of exposure. To evaluate such a possibility, we studied a set of cytosolic and microsomal GST isoform-specific expression profiles in the zebrafish embryos after exposure to atrazine, a widely used herbicide. Expression of the GST isoforms was compared with that of CYP genes involved in the phase I of xenobiotic metabolism and antioxidant enzyme (AOE) genes. Using quantitative real-time PCR, we showed dynamic changes in the expressional pattern of twenty GST isoforms, cyp1a, cyp3a65, ahr2, and four AOEs in early development of zebrafish. Acute (48 and 72 h) exposure of 24 h-old embryos to atrazine, from environmentally relevant (0.005 mg/L) to high (40 mg/L) concentrations, caused a variety of transient, albeit minor changes (GST isoforms, ahr2 and AOE genes response. However, expression of cyp1a and cyp3a65 mRNA was markedly and consistently induced by high doses of atrazine (5 and 40 mg/L). In summary, an analysis of the response of multiple systems in the zebrafish embryos provided a comprehensive understanding of atrazine toxicity and its potential impact on biological processes. PMID:25158112

  7. Identification of a novel ZIC3 isoform and mutation screening in patients with heterotaxy and congenital heart disease.

    Directory of Open Access Journals (Sweden)

    James E J Bedard

    Full Text Available Patients with heterotaxy have characteristic cardiovascular malformations, abnormal arrangement of their visceral organs, and midline patterning defects that result from abnormal left-right patterning during embryogenesis. Loss of function of the transcription factor ZIC3 causes X-linked heterotaxy and isolated congenital heart malformations and represents one of the few known monogenic causes of congenital heart disease. The birth incidence of heterotaxy-spectrum malformations is significantly higher in males, but our previous work indicated that mutations within ZIC3 did not account for the male over-representation. Therefore, cross species comparative sequence alignment was used to identify a putative novel fourth exon, and the existence of a novel alternatively spliced transcript was confirmed by amplification from murine embryonic RNA and subsequent sequencing. This transcript, termed Zic3-B, encompasses exons 1, 2, and 4 whereas Zic3-A encompasses exons 1, 2, and 3. The resulting protein isoforms are 466 and 456 amino acid residues respectively, sharing the first 407 residues. Importantly, the last two amino acids in the fifth zinc finger DNA binding domain are altered in the Zic3-B isoform, indicating a potential functional difference that was further evaluated by expression, subcellular localization, and transactivation analyses. The temporo-spatial expression pattern of Zic3-B overlaps with Zic3-A in vivo, and both isoforms are localized to the nucleus in vitro. Both isoforms can transcriptionally activate a Gli binding site reporter, but only ZIC3-A synergistically activates upon co-transfection with Gli3, suggesting that the isoforms are functionally distinct. Screening 109 familial and sporadic male heterotaxy cases did not identify pathogenic mutations in the newly identified fourth exon and larger studies are necessary to establish the importance of the novel isoform in human disease.

  8. Glycogen synthase isoforms in Synechocystis sp. PCC6803: identification of different roles to produce glycogen by targeted mutagenesis.

    Directory of Open Access Journals (Sweden)

    Sang-Ho Yoo

    Full Text Available Synechocystis sp. PCC6803 belongs to cyanobacteria which carry out photosynthesis and has recently become of interest due to the evolutionary link between bacteria and plant species. Similar to other bacteria, the primary carbohydrate storage source of Synechocystis sp. PCC6803 is glycogen. While most bacteria are not known to have any isoforms of glycogen synthase, analysis of the genomic DNA sequence of Synechocystis sp. PCC6803 predicts that this strain encodes two isoforms of glycogen synthase (GS for synthesizing glycogen structure. To examine the functions of the putative GS genes, each gene (sll1393 or sll0945 was disrupted by double cross-over homologous recombination. Zymogram analysis of the two GS disruption mutants allowed the identification of a protein band corresponding to each GS isoform. Results showed that two GS isoforms (GSI and GSII are present in Synechocystis sp. PCC6803, and both are involved in glycogen biosynthesis with different elongation properties: GSI is processive and GSII is distributive. Total GS activities in the mutant strains were not affected and were compensated by the remaining isoform. Analysis of the branch-structure of glycogen revealed that the sll1393- mutant (GSI- produced glycogen containing more intermediate-length chains (DP 8-18 at the expense of shorter and longer chains compared with the wild-type strain. The sll0945- mutant (GSII- produced glycogen similar to the wild-type, with only a slightly higher proportion of short chains (DP 4-11. The current study suggests that GS isoforms in Synechocystis sp. PCC6803 have different elongation specificities in the biosynthesis of glycogen, combined with ADP-glucose pyrophosphorylase and glycogen branching enzyme.

  9. Molecular Analysis of Collagen XVIII Reveals Novel Mutations, Presence of a Third Isoform, and Possible Genetic Heterogeneity in Knobloch Syndrome

    Science.gov (United States)

    Suzuki, O. T.; Sertié, A. L.; Der Kaloustian, V. M.; Kok, F.; Carpenter, M.; Murray, J.; Czeizel, A. E.; Kliemann, S. E.; Rosemberg, S.; Monteiro, M.; Olsen, B. R.; Passos-Bueno, M. R.

    2002-01-01

    Knobloch syndrome (KS) is a rare disease characterized by severe ocular alterations, including vitreoretinal degeneration associated with retinal detachment and occipital scalp defect. The responsible gene, COL18A1, has been mapped to 21q22.3, and, on the basis of the analysis of one family, we have demonstrated that a mutation affecting only one of the three COL18A1 isoforms causes this phenotype. We report here the results of the screening of both the entire coding region and the exon-intron boundaries of the COL18A1 gene (which includes 43 exons), in eight unrelated patients with KS. Besides 20 polymorphic changes, we identified 6 different pathogenic changes in both alleles of five unrelated patients with KS (three compound heterozygotes and two homozygotes). All are truncating mutations leading to deficiency of one or all collagen XVIII isoforms and endostatin. We have verified that, in exon 41, the deletion c3514-3515delCT, found in three unrelated alleles, is embedded in different haplotypes, suggesting that this mutation has occurred more than once. In addition, our results provide evidence of nonallelic genetic heterogeneity in KS. We also show that the longest human isoform (NC11-728) is expressed in several tissues (including the human eye) and that lack of either the short variant or all of the collagen XVIII isoforms causes similar phenotypes but that those patients who lack all forms present more-severe ocular alterations. Despite the small sample size, we found low endostatin plasma levels in those patients with mutations leading to deficiency of all isoforms; in addition, it seems that absence of all collagen XVIII isoforms causes predisposition to epilepsy. PMID:12415512

  10. Expression of the human apoE2 isoform in adipocytes: altered cellular processing and impaired adipocyte lipogenesis

    OpenAIRE

    Huang, Zhi H.; Maeda, Nobuyo; Mazzone, Theodore

    2011-01-01

    Expression of apoE in adipocytes has been shown to have an important role in modulating adipocyte triglyceride (TG) metabolism and gene expression that is independent of circulating and extracellular apoE. The impact of adipocyte expression of common human apoE isoforms was evaluated using adipocytes harvested from human apoE2, -3, and -4 knock-in mice. Expression of the apoE2 isoform was associated with an increase in adipocyte apoE gene expression and apoE synthesis. Newly synthesized apoE2...

  11. Specific T-type calcium channel isoforms are associated with distinct burst phenotypes in deep cerebellar nuclear neurons

    OpenAIRE

    Molineux, Michael L.; McRory, John E.; McKay, Bruce E.; Hamid, Jawed; Mehaffey, W. Hamish; Rehak, Renata; Snutch, Terrance P; Gerald W Zamponi; Turner, Ray W

    2006-01-01

    T-type calcium channels are thought to transform neuronal output to a burst mode by generating low voltage-activated (LVA) calcium currents and rebound burst discharge. In this study we assess the expression pattern of the three different T-type channel isoforms (Cav3.1, Cav3.2, and Cav3.3) in cerebellar neurons and focus on their potential role in generating LVA spikes and rebound discharge in deep cerebellar nuclear (DCN) neurons. We detected expression of one or more Cav3 channel isoforms ...

  12. Complex p63 mRNA isoform expression patterns in squamous cell carcinoma of the head and neck

    DEFF Research Database (Denmark)

    Thurfjell, Niklas; Coates, Philip J; Uusitalo, Tony;

    2004-01-01

    -isoform expression patterns and proliferation, p53 status, or telomerase expression. All p63 isoforms could be identified in normal surface epithelium, and micro-dissection showed that the high levels present in basal layers were similar to those seen in tumour tissues. Thus, high-level expression...... of deltaNp63 in tumour cells may represent maintained expression by the basal cells from which the tumour arose, rather than representing a true over-expression of p63 during tumourigenesis. Tobacco usage, a genotoxic predisposing factor for SCCHN, had no effect on p63 expression in oral epithelium. Taken...

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

    OpenAIRE

    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...

  14. Point mutation in the NF2 gene of HEI-193 human schwannoma cells results in the expression of a merlin isoform with attenuated growth suppressive activity

    Energy Technology Data Exchange (ETDEWEB)

    Lepont, Pierig; Stickney, John T.; Foster, Lauren A.; Meng, Jin-Jun; Hennigan, Robert F. [Department of Cell and Cancer Biology, Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267-0521 (United States); Ip, Wallace [Department of Cell and Cancer Biology, Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267-0521 (United States)], E-mail: wallace.ip@uc.edu

    2008-01-01

    Neurofibromatosis type 2 (NF2) is a genetic disorder characterized by the formation of bilateral schwannomas of the eighth cranial nerve. Although the protein product of the NF2 gene (merlin) is a classical tumor suppressor, the mechanism by which merlin suppresses cell proliferation is not fully understood. The availability of isolated tumor cells would facilitate a better understanding of the molecular function of merlin, but primary schwannoma cells obtained from patients grow slowly and do not yield adequate numbers for biochemical analysis. In this study, we have examined the NF2 mutation in HEI-193 cells, an immortalized cell line derived from the schwannoma of an NF2 patient. Previous work showed that the NF2 mutation in HEI-193 cells causes a splicing defect in the NF2 transcript. We have confirmed this result and further identified the resultant protein product as an isoform of merlin previously designated as isoform 3. The level of isoform 3 proteins in HEI-193 cells is comparable to the levels of merlin isoforms 1 and 2 in normal human Schwann cells and several other immortalized cell lines. In contrast to many mutant forms of merlin, isoform 3 is as resistant to proteasomal degradation as isoforms 1 and 2 and can interact with each of these isoforms in vivo. Cell proliferation assays showed that, in NF2{sup -/-} mouse embryonic fibroblasts, exogenously expressed merlin isoform 3 does exhibit growth suppressive activity although it is significantly lower than that of identically expressed merlin isoform 1. These results indicate that, although HEI-193 cells have undetectable levels of merlin isoforms 1 and 2, they are, in fact, not a merlin-null model because they express the moderately active growth suppressive merlin isoform 3.

  15. Nitric oxide in the bovine oviduct: influence on contractile activity and nitric oxide synthase isoforms localization.

    Science.gov (United States)

    Yilmaz, O; Całka, J; Bukowski, R; Zalecki, M; Wasowicz, K; Jaroszewski, J J; Markiewicz, W; Bulbul, A; Ucar, M

    2012-04-15

    The oviducts of 64 Holstein cows in luteal (early I, early II and late) and follicular phases were evaluated to determine the protein expression and mRNA transcription of different nitric oxide synthase isoforms (eNOS, iNOS, nNOS) as well as the effect of nitric oxide (NO) on spontaneous contractility in vitro. The expression patterns of nitric oxide synthase (NOS) isoforms in isthmus and ampulla (n = 6 for each phase) were determined by immunohistochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. In the contractility studies, longitudinal and circular isolated strips of isthmus and ampulla (n = 10 for each phase) of oviducts located ipsilateral to the luteal structure or preovulatory follicle were treated as follows: a) L-arginine, an endogenous NO donor (10(-8) to 10(-3)m), b) N(ω)-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor (10(-5)m) and L-arginine (10(-3)m), c) methylene blue (MB), an inhibitor of soluble guanylate (10(-5)m) and L-arginine (10(-3)m) and d) sodium nitroprusside (SNP), an exogenous NO donor (10(-8) to 10(-4)m). Immunohistochemical evaluation revealed that endothelial NOS (eNOS) expression detected in epithelial layer of isthmus and ampulla was strong in early I luteal phase, moderate in follicular phase and weak in other phases. Neuronal NOS (nNOS) immunoreactivity was strong in isthmus and moderate in ampulla, and staining of nerve fibers was observed mostly in early I luteal and follicular phases. All eNOS, nNOS and inducible NOS (iNOS) isoforms were detected by RT-PCR. eNOS and iNOS proteins were evident, whereas nNOS was undetectable by Western blot analysis in the tissue examined. L-arginine applied alone or after L-NAME did not alter or increase the contractile tension of the strips in most tissues examined. However, L-arginine applied after MB increased contractile tension in the strips of ampulla and longitudinal isthmus from early I luteal phase and circular isthmus from

  16. Toll-Like Receptor 9 Alternatively Spliced Isoform Negatively Regulates TLR9 Signaling in Teleost Fish

    Science.gov (United States)

    Chen, Nai-Yu; Nagarajan, Govindarajulu; Chiou, Pinwen Peter

    2015-01-01

    Toll-like receptor 9 (TLR9) recognizes and binds unmethylated CpG motifs in DNA, which are found in the genomes of bacteria and DNA viruses. In fish, Tlr9 is highly diverse, with the number of introns ranging from 0 to 4. A fish Tlr9 gene containing two introns has been reported to express two alternatively spliced isoforms, namely gTLR9A (full-length) and gTLR9B (with a truncated Cʹ-terminal signal transducing domain), whose regulation and function remain unclear. Here, we report a unique regulatory mechanism of gTLR9 signaling in orange-spotted grouper (Epinephelus coioides), whose gTlr9 sequence also contains two introns. We demonstrated that the grouper gTlr9 gene indeed has the capacity to produce two gTLR9 isoforms via alternative RNA splicing. We found that gTLR9B could function as a negative regulator to suppress gTLR9 signaling as demonstrated by the suppression of downstream gene expression. Following stimulation with CpG oligodeoxynucleotide (ODN), gTLR9A and gTLR9B were observed to translocate into endosomes and co-localize with ODN and the adaptor protein gMyD88. Both gTLR9A and gTLR9B could interact with gMyD88; however, gTLR9B could not interact with downstream IRAK4 and TRAF6. Further analysis of the expression profile of gTlr9A and gTlr9B upon immune-stimulation revealed that the two isoforms were differentially regulated in a time-dependent manner. Overall, these data suggest that fish TLR9B functions as a negative regulator, and that its temporal expression is mediated by alternative RNA splicing. This has not been observed in mammalian TLR9s and might have been acquired relatively recently in the evolution of fish. PMID:25955250

  17. Toll-Like Receptor 9 Alternatively Spliced Isoform Negatively Regulates TLR9 Signaling in Teleost Fish.

    Directory of Open Access Journals (Sweden)

    Frank Fang-Yao Lee

    Full Text Available Toll-like receptor 9 (TLR9 recognizes and binds unmethylated CpG motifs in DNA, which are found in the genomes of bacteria and DNA viruses. In fish, Tlr9 is highly diverse, with the number of introns ranging from 0 to 4. A fish Tlr9 gene containing two introns has been reported to express two alternatively spliced isoforms, namely gTLR9A (full-length and gTLR9B (with a truncated C'-terminal signal transducing domain, whose regulation and function remain unclear. Here, we report a unique regulatory mechanism of gTLR9 signaling in orange-spotted grouper (Epinephelus coioides, whose gTlr9 sequence also contains two introns. We demonstrated that the grouper gTlr9 gene indeed has the capacity to produce two gTLR9 isoforms via alternative RNA splicing. We found that gTLR9B could function as a negative regulator to suppress gTLR9 signaling as demonstrated by the suppression of downstream gene expression. Following stimulation with CpG oligodeoxynucleotide (ODN, gTLR9A and gTLR9B were observed to translocate into endosomes and co-localize with ODN and the adaptor protein gMyD88. Both gTLR9A and gTLR9B could interact with gMyD88; however, gTLR9B could not interact with downstream IRAK4 and TRAF6. Further analysis of the expression profile of gTlr9A and gTlr9B upon immune-stimulation revealed that the two isoforms were differentially regulated in a time-dependent manner. Overall, these data suggest that fish TLR9B functions as a negative regulator, and that its temporal expression is mediated by alternative RNA splicing. This has not been observed in mammalian TLR9s and might have been acquired relatively recently in the evolution of fish.

  18. Functional analysis of Na+/K+-ATPase isoform distribution in rat ventricular myocytes.

    Science.gov (United States)

    Despa, Sanda; Bers, Donald M

    2007-07-01

    The Na(+)/K(+)-ATPase (NKA) is the main route for Na(+) extrusion from cardiac myocytes. Different NKA alpha-subunit isoforms are present in the heart. NKA-alpha1 is predominant, although there is a variable amount of NKA-alpha2 in adult ventricular myocytes of most species. It has been proposed that NKA-alpha2 is localized mainly in T-tubules (TT), where it could regulate local Na(+)/Ca(2+) exchange and thus cardiac myocyte Ca(2+). However, there is controversy as to where NKA-alpha1 vs. NKA-alpha2 are localized in ventricular myocytes. Here, we assess the TT vs. external sarcolemma (ESL) distribution functionally using formamide-induced detubulation of rat ventricular myocytes, NKA current (I(Pump)) measurements and the different ouabain sensitivity of NKA-alpha1 (low) and NKA-alpha2 (high) in rat heart. Ouabain-dependent I(Pump) inhibition in control myocytes indicates a high-affinity NKA isoform (NKA-alpha2, K(1/2) = 0.38 +/- 0.16 microM) that accounts for 29.5 +/- 1.3% of I(Pump) and a low-affinity isoform (NKA-alpha1, K(1/2) = 141 +/- 17 microM) that accounts for 70.5% of I(Pump). Detubulation decreased cell capacitance from 164 +/- 6 to 120 +/- 8 pF and reduced I(Pump) density from 1.24 +/- 0.05 to 1.02 +/- 0.05 pA/pF, indicating that the functional density of NKA is significantly higher in TT vs. ESL. In detubulated myocytes, NKA-alpha2 accounted for only 18.2 +/- 1.1% of I(Pump). Thus, approximately 63% of I(Pump) generated by NKA-alpha2 is from the TT (although TT are only 27% of the total sarcolemma), and the NKA-alpha2/NKA-alpha1 ratio in TT is significantly higher than in the ESL. The functional density of NKA-alpha2 is approximately 4.5 times higher in the T-tubules vs. ESL, whereas NKA-alpha1 is almost uniformly distributed between the TT and ESL. PMID:17392375

  19. In vivo generation of neurotoxic prion protein: role for hsp70 in accumulation of misfolded isoforms.

    Directory of Open Access Journals (Sweden)

    Pedro Fernandez-Funez

    2009-06-01

    Full Text Available Prion diseases are incurable neurodegenerative disorders in which the normal cellular prion protein (PrP(C converts into a misfolded isoform (PrP(Sc with unique biochemical and structural properties that correlate with disease. In humans, prion disorders, such as Creutzfeldt-Jakob disease, present typically with a sporadic origin, where unknown mechanisms lead to the spontaneous misfolding and deposition of wild type PrP. To shed light on how wild-type PrP undergoes conformational changes and which are the cellular components involved in this process, we analyzed the dynamics of wild-type PrP from hamster in transgenic flies. In young flies, PrP demonstrates properties of the benign PrP(C; in older flies, PrP misfolds, acquires biochemical and structural properties of PrP(Sc, and induces spongiform degeneration of brain neurons. Aged flies accumulate insoluble PrP that resists high concentrations of denaturing agents and contains PrP(Sc-specific conformational epitopes. In contrast to PrP(Sc from mammals, PrP is proteinase-sensitive in flies. Thus, wild-type PrP rapidly converts in vivo into a neurotoxic, protease-sensitive isoform distinct from prototypical PrP(Sc. Next, we investigated the role of molecular chaperones in PrP misfolding in vivo. Remarkably, Hsp70 prevents the accumulation of PrP(Sc-like conformers and protects against PrP-dependent neurodegeneration. This protective activity involves the direct interaction between Hsp70 and PrP, which may occur in active membrane microdomains such as lipid rafts, where we detected Hsp70. These results highlight the ability of wild-type PrP to spontaneously convert in vivo into a protease-sensitive isoform that is neurotoxic, supporting the idea that protease-resistant PrP(Sc is not required for pathology. Moreover, we identify a new role for Hsp70 in the accumulation of misfolded PrP. Overall, we provide new insight into the mechanisms of spontaneous accumulation of neurotoxic PrP and uncover

  20. In vivo analysis of Saccharomyces cerevisiae plasma membrane ATPase Pma1p isoforms with increased in vitro H+/ATP stoichiometry

    NARCIS (Netherlands)

    De Kok, S.; Yilmaz, D.; Daran, J.M.; Pronk, J.T.; Van Maris, A.J.A.

    2012-01-01

    Plasma membrane H+-ATPase isoforms with increased H+/ATP ratios represent a desirable asset in yeast metabolic engineering. In vivo proton coupling of two previously reported Pma1p isoforms (Ser800Ala, Glu803Gln) with increased in vitro H+/ATP stoichiometries was analysed by measuring biomass yields

  1. DMPD: The role of C/EBP isoforms in the control of inflammatory and native immunityfunctions. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 9792624 The role of C/EBP isoforms in the control of inflammatory and native immuni...tyfunctions. Poli V. J Biol Chem. 1998 Nov 6;273(45):29279-82. (.png) (.svg) (.html) (.csml) Show The role o...f C/EBP isoforms in the control of inflammatory and native immunityfunctions. PubmedID 9792624 Title The role

  2. Expression of 1N3R-Tau isoform inhibits cell proliferation by inducing S phase arrest in N2a cells.

    Directory of Open Access Journals (Sweden)

    Li Li

    Full Text Available Tau is a microtubule-associated protein implicated in neurodegenerative tauopathies. Six tau isoforms are generated from a single gene through alternative splicing of exons 2, 3 and 10 in human brain. Differential expression of tau isoforms has been detected in different brain areas, during neurodevelopment and in neurodegenerative disorders. However, the biological significance of different tau isoforms is not clear. Here, we investigated the individual effect of six different isoforms of tau on cell proliferation and the possible mechanisms by transient expression of eGFP-labeled tau isoform plasmid in N2a cells. Our study showed the transfection efficiency was comparable between different isoforms of tau by examining GFP expression. Compared with other isoforms, we found expression of 1N3R-tau significantly inhibited cell proliferation by Cell Counting Kit-8 assay and BrdU incorporation. Flow cytometry analysis further showed expression of 1N3R-tau induced S phase arrest. Compared with the longest isoform of tau, expression of 1N3R-tau induced cyclin E translocation from the nuclei to cytoplasm, while it did not change the level of cell cycle checkpoint proteins. These data indicate that 1N3R-tau inhibits cell proliferation through inducing S phase arrest.

  3. High fat/low carbohydrate diet attenuates left ventricular hypertrophy and prevents myosin heavy chain isoform switching induced by chronic hypertenstion

    Science.gov (United States)

    A switch in the expression of myosin heavy chain isoform (MHC) alpha to beta is observed with left ventricular hypertrophy (LVH) and heart failure. This switch is associated with a defect in myocardial energy production and contractile dysfunction. Similar MHC isoform profile is observed in the fe...

  4. The HER4 isoform JM-a/CYT2 relates to improved survival in bladder cancer patients but only if the estrogen receptor α is not expressed

    DEFF Research Database (Denmark)

    Munk, Mathias; Memon, Ashfaque; Poulsen, Steen S;

    2013-01-01

    Abstract Bladder cancer tumors expressing human epidermal growth factor receptor 4 (HER4) demonstrate improved patient survival. HER4 isoforms and estrogen receptor alpha (ER-α) can form chaperone complexes causing cell-proliferation. We wanted to explore if HER4 isoforms and ER-α could correlate...... to poor prognosis in bladder cancers. We developed mRNA assays for HER4 isoforms (JM-a, JM-b, CYT1, and CYT2) and for ER-α. Expression was analyzed in tumors from 85 bladder cancer patients and compared to overall survival (median follow-up of 5.1 years). ER-α was expressed in 38% (n = 32) of tumors...... but did not correlate to survival (p = 0.4698). HER4 was expressed in 42% (n = 36) and in all cases as the ER-α binding isoform JM-a. The JM-a isoform can be alternatively spliced to either a CYT1 isoform (JM-a/CYT1) or a CYT2 isoform (JM-a/CYT2). All HER4 expressing tumors expressed the JM-a/CYT2 isoform...

  5. Total and isoform-specific quantitative assessment of circulating Fibulin-1 using selected reaction monitoring mass spectrometry and time-resolved immunofluorometry

    DEFF Research Database (Denmark)

    Overgaard, Martin; Cangemi, Claudia; Jensen, Martin L;

    2015-01-01

    biomarker fibulin-1 and its circulating isoforms in human plasma. EXPERIMENTAL DESIGN:: We used bioinformatics analysis to predict total and isoform-specific tryptic peptides for absolute quantitation using SRM-MS. Fibulin-1 was quantitated in plasma by nanoflow-LC-SRM-MS in undepleted plasma and time...

  6. Effect of diet on ability of Vascular Endothelial Growth Factor A (VEGFA) isoforms to alter follicular progression in bovine ovarian cortical cultures

    Science.gov (United States)

    The objective of this study was to determine the effect of changes in diet on ability of VEGFA isoforms to alter follicle progression in bovine ovarian cortex cultures. Our hypothesis was that diet would affect the magnitude of VEGFA isoform actions on follicular development. Heifers (n = 30) receiv...

  7. Impact of resistance exercise during bed rest on skeletal muscle sarcopenia and myosin isoform distribution

    Science.gov (United States)

    Bamman, M. M.; Clarke, M. S.; Feeback, D. L.; Talmadge, R. J.; Stevens, B. R.; Lieberman, S. A.; Greenisen, M. C.

    1998-01-01

    Because resistance exercise (REx) and bed-rest unloading (BRU) are associated with opposing adaptations, our purpose was to test the efficacy of REx against the effects of 14 days of BRU on the knee-extensor muscle group. Sixteen healthy men were randomly assigned to no exercise (NoEx; n = 8) or REx (n = 8). REx performed five sets of leg press exercise with 80-85% of one repetition maximum (1 RM) every other day during BRU. Muscle samples were removed from the vastus lateralis muscle by percutaneous needle biopsy. Myofiber distribution was determined immunohistochemically with three monoclonal antibodies against myosin heavy chain (MHC) isoforms (I, IIa, IIx). MHC distribution was further assessed by quantitative gel electrophoresis. Dynamic 1-RM leg press and unilateral maximum voluntary isometric contraction (MVC) were determined. Maximal neural activation (root mean squared electromyogram) and rate of torque development (RTD) were measured during MVC. Reductions (P exercise for astronauts in microgravity.

  8. Nanoscale elucidation of Na,K-ATPase isoforms in dendritic spines

    CERN Document Server

    Liebmann, Thomas; Aperia, Anita; Brismar, Hjalmar

    2013-01-01

    The dimensions of neuronal synapses suggest that optical super-resolution imaging methods are necessary for thorough investigation of protein distributions and interactions. Nanoscopic evaluation of neuronal samples has presented practical hurdles, but advancing methods are making synaptic protein topology and quantification measurements feasible. This work explores the application of Photoactivated Localization Microscopy (PALM) pointilistic super-resolution imaging for investigation of the membrane bound sodium pump, the Na,K-ATPase, in matured neurons. Two isoforms of the sodium pump (ATP1a1 and ATP1a3) were studied in cultured neurons using the PALM-compatible fluorescent proteins PAGFP and mEos. Nanoscopic imaging reveals a compartmentalized distribution of sodium pumps in dendritic spines. Several nanoclusters of pumps are typically found in the spine head and relatively few in the spine neck. The density of sodium pumps was estimated from a quantification of detected single molecules to 600-800 pump co...

  9. Nonmetabolic functions of pyruvate kinase isoform M2 in controlling cell cycle progression and tumorigenesis

    Institute of Scientific and Technical Information of China (English)

    Zhimin Lu

    2012-01-01

    Pyruvate kinase catalyzes the rate-limiting final step of glycolysis,generating adenosine triphosphate (ATP) and pyruvate.The M2 tumor-specific isoform of pyruvate kinase (PKM2) promotes glucose uptake and lactate production in the presence of oxygen,known as aerobic glycolysis or the Warburg effect.As recently reported in Nature,PKM2,besides its metabolic function,has a nonmetabolic function in the direct control of cell cycle progression by activating β-catenin and inducing expression of the β-catenin downstream gene CCND1 (encoding for cyclin D1).This nonmetabolic function of PKM2 is essential for epidermal growth factor receptor (EGFR) activation-induced tumorigenesis.

  10. Expression of glutamine transporter isoforms in cerebral cortex of rats with chronic hepatic encephalopathy

    DEFF Research Database (Denmark)

    Leke, Renata; Escobar, Thayssa D.C.; Rama Rao, Kakulavarapu V.;

    2015-01-01

    Hepatic encephalopathy (HE) is a neuropsychiatric disorder that occurs due to acute and chronic liver diseases, the hallmark of which is the increased levels of ammonia and subsequent alterations in glutamine synthesis, i.e. conditions associated with the pathophysiology of HE. Under physiological...... conditions, glutamine is fundamental for replenishment of the neurotransmitter pools of glutamate and GABA. The different isoforms of glutamine transporters play an important role in the transfer of this amino acid between astrocytes and neurons. A disturbance in the GABA biosynthetic pathways has been...... described in bile duct ligated (BDL) rats, a well characterized model of chronic HE. Considering that glutamine is important for GABA biosynthesis, altered glutamine transport and the subsequent glutamate/GABA–glutamine cycle efficacy might influence these pathways. Given this potential outcome, the aim...

  11. Probing polypeptide GalNAc-transferase isoform substrate specificities by in vitro analysis

    DEFF Research Database (Denmark)

    Kong, Yun; Joshi, Hiren J; Schjoldager, Katrine Ter-Borch Gram;

    2015-01-01

    N-acetylgalactosaminyltransferase (GalNAc)-type (mucin-type) O-glycosylation is an abundant and highly diverse modification of proteins. This type of O-glycosylation is initiated in the Golgi by a large family of up to 20 homologous polypeptide GalNAc-T isoenzymes that transfer GalNAc to Ser, Thr...... and possibly Tyr residues. These GalNAc residues are then further elongated by a large set of glycosyltransferases to build a variety of complex O-glycan structures. What determines O-glycan site occupancy is still poorly understood, although it is clear that the substrate specificities of individual...... isoenzymes and the repertoire of GalNAc-Ts in cells are key parameters. The GalNAc-T isoenzymes are differentially expressed in cells and tissues in principle allowing cells to produce unique O-glycoproteomes dependent on the specific subset of isoforms present. In vitro analysis of acceptor peptide...

  12. Differentiation of lethal and non lethal prostate cancer:PSA and PSA isoforms and kinetics

    Institute of Scientific and Technical Information of China (English)

    H Ballentine Carter

    2012-01-01

    Prostate-specific antigen (PSA) testing for the early diagnosis of prostate cancer has led to a decrease in cancer mortality.However,the high prevalence of low-grade prostate cancer and its long natural history,competing causes of death in older men and treatment patterns of prostate cancer,have led to dramatic overtreatment of the disease.Improved markers of prostate cancer lethality are needed to reduce the overtreatment of prostate cancer that leads to a reduced quality of life without extending life for a high proportion of men.The PSA level prior to treatment is routinely used in multivariable models to predict prostate cancer aggressiveness.PSA isoforms and PSA kinetics have been associated with more aggressive phenotypes,but are not routinely employed as part of prediction tools prior to treatment.PSA kinetics is a valuable marker of lethality post treatment and routinely used in determining the need for salvage therapy.

  13. Molecular cloning and characterization of rat brain 2',3'-cyclic nucleotide 3'-phosphodiesterase isoform 2.

    Science.gov (United States)

    Gravel, M; DeAngelis, D; Braun, P E

    1994-06-15

    We have isolated a cDNA coding for the larger isoform of the rat brain 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP2), a protein associated with myelination in the central nervous system (CNS). The complete 420 amino acid sequence was deduced from the nucleotide sequence of the cDNA. Sequence comparisons show that rat CNP shares 96% homology with mouse, 84% with bovine, and 86% with human CNP. Errors in the published sequence of rat CNP1 have now been corrected. Comparisons with other proteins reveal several interesting conserved motifs, including two leucine repeat heptads, and two consensus motifs for phosphorylation in the N-terminal domain of CNP2. PMID:7932861

  14. Functional refolding and characterization of two Tom40 isoforms from human mitochondria.

    Science.gov (United States)

    Mager, Frauke; Gessmann, Dennis; Nussberger, Stephan; Zeth, Kornelius

    2011-07-01

    Tom40 proteins represent an essential class of molecules which facilitate translocation of unfolded proteins from the cytosol into the mitochondrial intermembrane space. They are part of a high-molecular mass complex that forms the protein-conducting channel in outer mitochondrial membranes. This study concerns the recombinant expression, purification and folding of amino-terminally truncated variants of the two human Tom40 isoforms for structural biology experiments. Both CD and FTIR secondary structure analysis revealed a dominant beta-sheet structure and a short alpha-helical part for both proteins together with a high thermal stability. Two secondary structure elements can be denatured independently. Reconstitution of the recombinant protein into planar lipid bilayers demonstrated ion channel activity similar to Tom40 purified from Neurospora crassa mitochondrial membranes, but conductivity fingerprints differ from the structurally closely related VDAC proteins.

  15. Direct binding and activation of protein kinase C isoforms by steroid hormones.

    LENUS (Irish Health Repository)

    Alzamora, Rodrigo

    2008-10-01

    The non-genomic action of steroid hormones regulates a wide variety of cellular responses including regulation of ion transport, cell proliferation, migration, death and differentiation. In order to achieve such plethora of effects steroid hormones utilize nearly all known signal transduction pathways. One of the key signalling molecules regulating the non-genomic action of steroid hormones is protein kinase C (PKC). It is thought that rapid action of steroids hormones results from the activation of plasma membrane receptors; however, their molecular identity remains elusive. In recent years, an increasing number of studies have pointed at the selective binding and activation of specific PKC isoforms by steroid hormones. This has led to the hypothesis that PKC could act as a receptor as well as a transducer of the non-genomic effects of these hormones. In this review we summarize the current knowledge of the direct binding and activation of PKC by steroid hormones.

  16. HER4 and its cytoplasmic isoforms are associated with progression-free survival of malignant melanoma

    DEFF Research Database (Denmark)

    Nielsen, Trine O; Poulsen, Steen Seier; Journe, Fabrice;

    2014-01-01

    RNA quantification was carried out of all four EGF receptors (EGFR, HER2, HER3, and HER4) and the HER4 cytoplasmic isoforms in lymph node metastases from patients with malignant melanoma. We related their expression to progression of the disease. HER4 expression was found to be an indicator of short progression......-free survival (P=0.0340). Interestingly, of the two cytoplasmic splice variants of HER4, the association of CYT1 (P=0.0176) with progression-free survival was more pronounced than that for CYT2 (P=0.0458). Also, HER3 was associated with progression-free survival (P=0.0169), whereas no association was found for...

  17. Impact of resistance exercise during bed rest on skeletal muscle sarcopenia and myosin isoform distribution

    Science.gov (United States)

    Bamman, M. M.; Clarke, M. S.; Feeback, D. L.; Talmadge, R. J.; Stevens, B. R.; Lieberman, S. A.; Greenisen, M. C.

    1998-01-01

    Because resistance exercise (REx) and bed-rest unloading (BRU) are associated with opposing adaptations, our purpose was to test the efficacy of REx against the effects of 14 days of BRU on the knee-extensor muscle group. Sixteen healthy men were randomly assigned to no exercise (NoEx; n = 8) or REx (n = 8). REx performed five sets of leg press exercise with 80-85% of one repetition maximum (1 RM) every other day during BRU. Muscle samples were removed from the vastus lateralis muscle by percutaneous needle biopsy. Myofiber distribution was determined immunohistochemically with three monoclonal antibodies against myosin heavy chain (MHC) isoforms (I, IIa, IIx). MHC distribution was further assessed by quantitative gel electrophoresis. Dynamic 1-RM leg press and unilateral maximum voluntary isometric contraction (MVC) were determined. Maximal neural activation (root mean squared electromyogram) and rate of torque development (RTD) were measured during MVC. Reductions (P < 0.05) in type I (15%) and type II (17%) myofiber cross-sectional areas were found in NoEx but not in REx. Electrophoresis revealed no changes in MHC isoform distribution. The percentage of type IIx myofibers decreased (P < 0.05) in REx from 9 to 2% and did not change in NoEx. 1 RM was reduced (P < 0.05) by 9% in NoEx but was unchanged in REx. MVC fell by 15 and 13% in NoEx and REx, respectively. The agonist-to-antagonist root mean squared electromyogram ratio decreased (P < 0.05) 19% in REx. RTD slowed (P < 0.05) by 54% in NoEx only. Results indicate that REx prevented BRU-induced myofiber atrophy and also maintained training-specific strength. Unlike spaceflight, BRU did not induce shifts in myosin phenotype. The reported benefits of REx may prove useful in prescribing exercise for astronauts in microgravity.

  18. N(ε)-Carboxymethyl Modification of Lysine Residues in Pathogenic Prion Isoforms.

    Science.gov (United States)

    Choi, Yeong-Gon; Shin, Hae-Young; Kim, Jae-Il; Choi, Eun-Kyoung; Carp, Richard I; Kim, Yong-Sun

    2016-07-01

    The most prominent hallmark of prion diseases is prion protein conversion and the subsequent deposition of the altered prions, PrP(Sc), at the pathological sites of affected individuals, particularly in the brain. A previous study has demonstrated that the N-terminus of the pathogenic prion isoform (PrP(Sc)) is modified with advanced glycation end products (AGEs), most likely at one or more of the three Lys residues (positions 23, 24, and 27) in the N-terminus (23KKRPKP28). The current study investigated whether N(ε)-(carboxymethyl)lysine (CML), a major AGE form specific to Lys residues produced by nonenzymatic glycation, is an AGE adduct of the N-terminus of PrP(Sc). We show that CML is linked to at least one Lys residue at the N-terminus of PrP(Sc) in 263K prion-infected hamster brains and at least one of the eight Lys residues (positions 101, 104, 106, 110, 185, 194, 204, and 220) in the proteinase K (PK)-resistant core region of PrP(Sc). The nonenzymatic glycation of the Lys residue(s) of PrP(Sc) with CML likely occurs in the widespread prion-deposit areas within infected brains, particularly in some of the numerous tyrosine hydroxylase-positive thalamic and hypothalamic nuclei. CML glycation does not occur in PrP(C) but is seen in the pathologic PrP(Sc) isoform. Furthermore, the modification of PrP(Sc) with CML may be closely involved in prion propagation and deposition in pathological brain areas. PMID:25983034

  19. Expression of CD150 in tumors of the central nervous system: identification of a novel isoform.

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    Olga Romanets-Korbut

    Full Text Available CD150 (IPO3/SLAM belongs to the SLAM family of receptors and serves as a major entry receptor for measles virus. CD150 is expressed on normal and malignant cells of the immune system. However, little is known about its expression outside the hematopoietic system, especially tumors of the central nervous system (CNS. Although CD150 was not found in different regions of normal brain tissues, our immunohistochemical study revealed its expression in 77.6% of human CNS tumors, including glioblastoma, anaplastic astrocytoma, diffuse astrocytoma, ependymoma, and others. CD150 was detected in the cytoplasm, but not on the cell surface of glioma cell lines, and it was colocalized with the endoplasmic reticulum and Golgi complex markers. In addition to the full length mRNA of the mCD150 splice isoform, in glioma cells we found a highly expressed novel CD150 transcript (nCD150, containing an 83 bp insert. The insert is derived from a previously unrecognized exon designated Cyt-new, which is located 510 bp downstream of the transmembrane region exon, and is a specific feature of primate SLAMF1. Both mCD150 and nCD150 cDNA variants did not contain any mutations and had the leader sequence. The nCD150 transcript was also detected in normal and malignant B lymphocytes, primary T cells, dendritic cells and macrophages; however, in glioma cells nCD150 was found to be the predominant CD150 isoform. Similarly to mCD150, cell surface expression of nCD150 allows wild type measles virus entry to the cell. Our data indicate that CD150 expression in CNS tumors can be considered a new diagnostic marker and potential target for novel therapeutic approaches.

  20. Isoform 1 of TPD52 (PC-1) promotes neuroendocrine transdifferentiation in prostate cancer cells

    KAUST Repository

    Moritz, Tom

    2016-02-05

    The tumour protein D52 isoform 1 (PC-1), a member of the tumour protein D52 (TPD52) protein family, is androgen-regulated and prostate-specific expressed. Previous studies confirmed that PC-1 contributes to malignant progression in prostate cancer with an important role in castration-resistant stage. In the present work, we identified its impact in mechanisms leading to neuroendocrine (NE) transdifferentiation. We established for long-term PC-1 overexpression an inducible expression system derived from the prostate carcinoma cell line LNCaP. We observed that PC-1 overexpression itself initiates characteristics of neuroendocrine cells, but the effect was much more pronounced in the presence of the cytokine interleukin-6 (IL-6). Moreover, to our knowledge, this is the first report that treatment with IL-6 leads to a significant upregulation of PC-1 in LNCaP cells. Other TPD52 isoforms were not affected. Proceeding from this result, we conclude that PC-1 overexpression enhances the IL-6-mediated differentiation of LNCaP cells into a NE-like phenotype, noticeable by morphological changes and increased expression of typical NE markers, like chromogranin A, synaptophysin or beta-3 tubulin. Immunofluorescent staining of IL-6-treated PC-1-overexpressing LNCaP cells indicates a considerable PC-1 accumulation at the end of the long-branched neuron-like cell processes, which are typically formed by NE cells. Additionally, the experimentally initiated NE transdifferentiation correlates with the androgen receptor status, which was upregulated additively. In summary, our data provide evidence for an involvement of PC-1 in NE transdifferentiation, frequently associated with castration resistance, which is a major therapeutic challenge in the treatment of advanced prostate cancer.

  1. Diverging regulation of pyruvate dehydrogenase kinase isoform gene expression in cultured human muscle cells.

    Science.gov (United States)

    Abbot, Emily L; McCormack, James G; Reynet, Christine; Hassall, David G; Buchan, Kevin W; Yeaman, Stephen J

    2005-06-01

    The pyruvate dehydrogenase complex occupies a central and strategic position in muscle intermediary metabolism and is primarily regulated by phosphorylation/dephosphorylation. The identification of multiple isoforms of pyruvate dehydrogenase kinase (PDK1-4) and pyruvate dehydrogenase phosphatase (PDP1-2) has raised intriguing new possibilities for chronic pyruvate dehydrogenase complex control. Experiments to date suggest that PDK4 is the major isoenzyme responsible for changes in pyruvate dehydrogenase complex activity in response to various different metabolic conditions. Using a cultured human skeletal muscle cell model system, we found that expression of both PDK2 and PDK4 mRNA is upregulated in response to glucose deprivation and fatty acid supplementation, the effects of which are reversed by insulin treatment. In addition, insulin directly downregulates PDK2 and PDK4 mRNA transcript abundance via a phosphatidylinositol 3-kinase-dependent pathway, which may involve glycogen synthase kinase-3 but does not utilize the mammalian target of rapamycin or mitogen-activated protein kinase signalling pathways. In order to further elucidate the regulation of PDK, the role of the peroxisome proliferators-activated receptors (PPAR) was investigated using highly potent subtype selective agonists. PPARalpha and PPARdelta agonists were found to specifically upregulate PDK4 mRNA expression, whereas PPARgamma activation selectively decreased PDK2 mRNA transcript abundance. PDP1 mRNA expression was unaffected by all conditions analysed. These results suggest that in human muscle, hormonal and nutritional conditions may control PDK2 and PDK4 mRNA expression via a common signalling mechanism. In addition, PPARs appear to independently regulate specific PDK isoform transcipt levels, which are likely to impart important metabolic mediation of fuel utilization by the muscle. PMID:15955060

  2. Plasma proteome changes in cardiovascular disease patients: novel isoforms of apolipoprotein A1

    Directory of Open Access Journals (Sweden)

    Oravec Milan

    2011-06-01

    Full Text Available Abstract Background The aim of this proteomic study was to look for changes taking place in plasma proteomes of patients with acute myocardial infarction (AMI, unstable angina pectoris (UAP, and stable angina pectoris (SAP. Methods Depleted plasma proteins were separated by 2D SDS-PAGE (pI 4-7, and proteomes were compared using Progenesis SameSpots statistical software. Proteins were identified by nanoLC-MS/MS. Proteins were quantified using commercial kits. Apolipoprotein A1 was studied using 1D and 2D SDS-PAGE, together with western blotting. Results Reciprocal comparison revealed 46 unique, significantly different spots; proteins in 34 spots were successfully identified and corresponded to 38 different proteins. Discrete comparisons of patient groups showed 45, 41, and 8 significantly different spots when AMI, UAP, and SAP were compared with the control group. On the basis of our proteomic data, plasma levels of two of them, alpha-1 microglobulin and vitamin D-binding protein, were determined. The data, however, failed to prove the proteins to be suitable markers or risk factors in the studied groups. The plasma level and isoform representation of apolipoprotein A1 were also estimated. Using 1D and 2D SDS-PAGE, together with western blotting, we observed extra high-molecular weight apolipoprotein A1 fractions presented only in the patient groups, indicating that the novel high-molecular weight isoforms of apolipoprotein A1 may be potential new markers or possible risk factors of cardiovascular disease. Conclusion The reported data show plasma proteome changes in patients with AMI, UAP, and SAP. We propose some apolipoprotein A1 fractions as a possible new disease-associated marker of cardiovascular disorders.

  3. Imaging single retrovirus entry through alternative receptor isoforms and intermediates of virus-endosome fusion.

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    Naveen K Jha

    Full Text Available A large group of viruses rely on low pH to activate their fusion proteins that merge the viral envelope with an endosomal membrane, releasing the viral nucleocapsid. A critical barrier to understanding these events has been the lack of approaches to study virus-cell membrane fusion within acidic endosomes, the natural sites of virus nucleocapsid capsid entry into the cytosol. Here we have investigated these events using the highly tractable subgroup A avian sarcoma and leukosis virus envelope glycoprotein (EnvA-TVA receptor system. Through labeling EnvA pseudotyped viruses with a pH-sensitive fluorescent marker, we imaged their entry into mildly acidic compartments. We found that cells expressing the transmembrane receptor (TVA950 internalized the virus much faster than those expressing the GPI-anchored receptor isoform (TVA800. Surprisingly, TVA800 did not accelerate virus uptake compared to cells lacking the receptor. Subsequent steps of virus entry were visualized by incorporating a small viral content marker that was released into the cytosol as a result of fusion. EnvA-dependent fusion with TVA800-expressing cells occurred shortly after endocytosis and delivery into acidic endosomes, whereas fusion of viruses internalized through TVA950 was delayed. In the latter case, a relatively stable hemifusion-like intermediate preceded the fusion pore opening. The apparent size and stability of nascent fusion pores depended on the TVA isoforms and their expression levels, with TVA950 supporting more robust pores and a higher efficiency of infection compared to TVA800. These results demonstrate that surface receptor density and the intracellular trafficking pathway used are important determinants of efficient EnvA-mediated membrane fusion, and suggest that early fusion intermediates play a critical role in establishing low pH-dependent virus entry from within acidic endosomes.

  4. Identification and Analysis of the Role of Superoxide Dismutases Isoforms in the Pathogenesis of Paracoccidioides spp.

    Science.gov (United States)

    Tamayo, Diana; Muñoz, José F.; Lopez, Ángela; Urán, Martha; Herrera, Juan; Borges, Clayton L.; Restrepo, Ángela; Soares, Celia M.; Taborda, Carlos P.; Almeida, Agostinho J.; McEwen, Juan G.; Hernández, Orville

    2016-01-01

    The ability of Paracoccidioides to defend itself against reactive oxygen species (ROS) produced by host effector cells is a prerequisite to survive. To counteract these radicals, Paracoccidioides expresses, among different antioxidant enzymes, superoxide dismutases (SODs). In this study, we identified six SODs isoforms encoded by the Paracoccidioides genome. We determined gene expression levels of representative isolates of the phylogenetic lineages of Paracoccidioides spp. (S1, PS2, PS3 and Pb01-like) using quantitative RT-PCR. Assays were carried out to analyze SOD gene expression of yeast cells, mycelia cells, the mycelia-to-yeast transition and the yeast-to-mycelia germination, as well as under treatment with oxidative agents and during interaction with phagocytic cells. We observed an increased expression of PbSOD1 and PbSOD3 during the transition process, exposure to oxidative agents and interaction with phagocytic cells, suggesting that these proteins could assist in combating the superoxide radicals generated during the host-pathogen interaction. Using PbSOD1 and PbSOD3 knockdown strains we showed these genes are involved in the response of the fungus against host effector cells, particularly the oxidative stress response, and in a mouse model of infection. Protein sequence analysis together with functional analysis of knockdown strains seem to suggest that PbSOD3 expression is linked with a pronounced extracellular activity while PbSOD1 seems more related to intracellular requirements of the fungus. Altogether, our data suggests that P. brasiliensis actively responds to the radicals generated endogenously during metabolism and counteracts the oxidative burst of immune cells by inducing the expression of SOD isoforms. PMID:26963091

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

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    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.

  6. Identification and Analysis of the Role of Superoxide Dismutases Isoforms in the Pathogenesis of Paracoccidioides spp.

    Directory of Open Access Journals (Sweden)

    Diana Tamayo

    2016-03-01

    Full Text Available The ability of Paracoccidioides to defend itself against reactive oxygen species (ROS produced by host effector cells is a prerequisite to survive. To counteract these radicals, Paracoccidioides expresses, among different antioxidant enzymes, superoxide dismutases (SODs. In this study, we identified six SODs isoforms encoded by the Paracoccidioides genome. We determined gene expression levels of representative isolates of the phylogenetic lineages of Paracoccidioides spp. (S1, PS2, PS3 and Pb01-like using quantitative RT-PCR. Assays were carried out to analyze SOD gene expression of yeast cells, mycelia cells, the mycelia-to-yeast transition and the yeast-to-mycelia germination, as well as under treatment with oxidative agents and during interaction with phagocytic cells. We observed an increased expression of PbSOD1 and PbSOD3 during the transition process, exposure to oxidative agents and interaction with phagocytic cells, suggesting that these proteins could assist in combating the superoxide radicals generated during the host-pathogen interaction. Using PbSOD1 and PbSOD3 knockdown strains we showed these genes are involved in the response of the fungus against host effector cells, particularly the oxidative stress response, and in a mouse model of infection. Protein sequence analysis together with functional analysis of knockdown strains seem to suggest that PbSOD3 expression is linked with a pronounced extracellular activity while PbSOD1 seems more related to intracellular requirements of the fungus. Altogether, our data suggests that P. brasiliensis actively responds to the radicals generated endogenously during metabolism and counteracts the oxidative burst of immune cells by inducing the expression of SOD isoforms.

  7. Identification and Analysis of the Role of Superoxide Dismutases Isoforms in the Pathogenesis of Paracoccidioides spp.

    Science.gov (United States)

    Tamayo, Diana; Muñoz, José F; Lopez, Ángela; Urán, Martha; Herrera, Juan; Borges, Clayton L; Restrepo, Ángela; Soares, Celia M; Taborda, Carlos P; Almeida, Agostinho J; McEwen, Juan G; Hernández, Orville

    2016-03-01

    The ability of Paracoccidioides to defend itself against reactive oxygen species (ROS) produced by host effector cells is a prerequisite to survive. To counteract these radicals, Paracoccidioides expresses, among different antioxidant enzymes, superoxide dismutases (SODs). In this study, we identified six SODs isoforms encoded by the Paracoccidioides genome. We determined gene expression levels of representative isolates of the phylogenetic lineages of Paracoccidioides spp. (S1, PS2, PS3 and Pb01-like) using quantitative RT-PCR. Assays were carried out to analyze SOD gene expression of yeast cells, mycelia cells, the mycelia-to-yeast transition and the yeast-to-mycelia germination, as well as under treatment with oxidative agents and during interaction with phagocytic cells. We observed an increased expression of PbSOD1 and PbSOD3 during the transition process, exposure to oxidative agents and interaction with phagocytic cells, suggesting that these proteins could assist in combating the superoxide radicals generated during the host-pathogen interaction. Using PbSOD1 and PbSOD3 knockdown strains we showed these genes are involved in the response of the fungus against host effector cells, particularly the oxidative stress response, and in a mouse model of infection. Protein sequence analysis together with functional analysis of knockdown strains seem to suggest that PbSOD3 expression is linked with a pronounced extracellular activity while PbSOD1 seems more related to intracellular requirements of the fungus. Altogether, our data suggests that P. brasiliensis actively responds to the radicals generated endogenously during metabolism and counteracts the oxidative burst of immune cells by inducing the expression of SOD isoforms. PMID:26963091

  8. Titin isoform switching is a major cardiac adaptive response in hibernating grizzly bears.

    Science.gov (United States)

    Nelson, O Lynne; Robbins, Charles T; Wu, Yiming; Granzier, Henk

    2008-07-01

    The hibernation phenomenon captures biological as well as clinical interests to understand how organs adapt. Here we studied how hibernating grizzly bears (Ursus arctos horribilis) tolerate extremely low heart rates without developing cardiac chamber dilation. We evaluated cardiac filling function in unanesthetized grizzly bears by echocardiography during the active and hibernating period. Because both collagen and titin are involved in altering diastolic function, we investigated both in the myocardium of active and hibernating grizzly bears. Heart rates were reduced from 84 beats/min in active bears to 19 beats/min in hibernating bears. Diastolic volume, stroke volume, and left ventricular ejection fraction were not different. However, left ventricular muscle mass was significantly lower (300 +/- 12 compared with 402 +/- 14 g; P = 0.003) in the hibernating bears, and as a result the diastolic volume-to-left ventricular muscle mass ratio was significantly greater. Early ventricular filling deceleration times (106.4 +/- 14 compared with 143.2 +/- 20 ms; P = 0.002) were shorter during hibernation, suggesting increased ventricular stiffness. Restrictive pulmonary venous flow patterns supported this conclusion. Collagen type I and III comparisons did not reveal differences between the two groups of bears. In contrast, the expression of titin was altered by a significant upregulation of the stiffer N2B isoform at the expense of the more compliant N2BA isoform. The mean ratio of N2BA to N2B titin was 0.73 +/- 0.07 in the active bears and decreased to 0.42 +/- 0.03 (P = 0.006) in the hibernating bears. The upregulation of stiff N2B cardiac titin is a likely explanation for the increased ventricular stiffness that was revealed by echocardiography, and we propose that it plays a role in preventing chamber dilation in hibernating grizzly bears. Thus our work identified changes in the alternative splicing of cardiac titin as a major adaptive response in hibernating grizzly

  9. Analysis of tarantula skeletal muscle protein sequences and identification of transcriptional isoforms

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    Yu Jun

    2009-03-01

    Full Text Available Abstract Background Tarantula has been used as a model system for studying skeletal muscle structure and function, yet data on the genes expressed in tarantula muscle are lacking. Results We constructed a cDNA library from Aphonopelma sp. (Tarantula skeletal muscle and got 2507 high-quality 5'ESTs (expressed sequence tags from randomly picked clones. EST analysis showed 305 unigenes, among which 81 had more than 2 ESTs. Twenty abundant unigenes had matches to skeletal muscle-related genes including actin, myosin, tropomyosin, troponin-I, T and C, paramyosin, muscle LIM protein, muscle protein 20, a-actinin and tandem Ig/Fn motifs (found in giant sarcomere-related proteins. Matches to myosin light chain kinase and calponin were also identified. These results support the existence of both actin-linked and myosin-linked regulation in tarantula skeletal muscle. We have predicted full-length as well as partial cDNA sequences both experimentally and computationally for myosin heavy and light chains, actin, tropomyosin, and troponin-I, T and C, and have deduced the putative peptides. A preliminary analysis of the structural and functional properties was also carried out. Sequence similarities suggested multiple isoforms of most myofibrillar proteins, supporting the generality of multiple isoforms known from previous muscle sequence studies. This may be related to a mix of muscle fiber types. Conclusion The present study serves as a basis for defining the transcriptome of tarantula skeletal muscle, for future in vitro expression of tarantula proteins, and for interpreting structural and functional observations in this model species.

  10. The β isoform of GSK3 mediates podocyte autonomous injury in proteinuric glomerulopathy.

    Science.gov (United States)

    Li, Changbin; Ge, Yan; Dworkin, Lance; Peng, Ai; Gong, Rujun

    2016-05-01

    Converging evidence points to glycogen synthase kinase (GSK) 3 as a key player in the pathogenesis of podocytopathy and proteinuria. However, it remains unclear if GSK3 is involved in podocyte autonomous injury in glomerular disease. In normal kidneys, the β isoform of GSK3 was found to be the major GSK3 expressed in glomeruli and intensely stained in podocytes. GSK3β expression in podocytes was markedly elevated in experimental or human proteinuric glomerulopathy. Podocyte-specific somatic ablation of GSK3β in adult mice attenuated proteinuria and ameliorated podocyte injury and glomerular damage in experimental adriamycin (ADR) nephropathy. Mechanistically, actin cytoskeleton integrity in podocytes was largely preserved in GSK3β knockout mice following ADR insult, concomitant with a correction of podocyte hypermotility and lessened phosphorylation and activation of paxillin, a focal adhesion-associated adaptor protein. In addition, GSK3β knockout diminished ADR-induced NFκB RelA/p65 phosphorylation selectively at serine 467; suppressed de novo expression by podocytes of NFκB-dependent podocytopathic mediators, including B7-1, cathepsin L, and MCP-1; but barely affected the induction of NFκB target pro-survival factors, such as Bcl-xL. Moreover, the ADR-elicited podocytopenia and podocyte death were significantly attenuated in GSK3β knockout mice, associated with protection against podocyte mitochondrial damage and reduced phosphorylation and activation of cyclophilin F, a structural component of mitochondria permeability transition pores. Overall, our findings suggest that the β isoform of GSK3 mediates autonomous podocyte injury in glomerulopathy by integrating multiple podocytopathic signalling pathways. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. PMID:26876299

  11. Comparison of α-amylase isoforms from the midgut of Cerambyx cerdo L. (Coleoptera: Cerambycidae larvae developed in the wild and on an artificial diet

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    Dojnov Biljana

    2010-01-01

    Full Text Available α-Amylase isoforms of Cerambyx cerdo larvae from the wild (ML and SL and reared in the laboratory (ADL were compared. Three amylase isoforms were presented in the SL and ML extracts while two isoforms were presented in the ADL according to zymogram after isoelectric focusing (IEF. All C. cerdo amylase isoforms were acidic proteins (pI < 3.5. Seven amylase isoforms (ACC 1-7 from the midgut of C. cerdo larvae were found in the ML midgut extract, six in the SL extract, and four in the ADL extract according to native PAGE zymogram. The ADL amylase had the highest activity. All crude midgut extracts of C. cerdo larvae were fractionated on a Superose 12 HR column. The molecular mass of the ACC was estimated to be 34 kDa. .

  12. Early detection of skeletal muscle injury by assay of creatine kinase MM isoforms in serum after acute exercise

    DEFF Research Database (Denmark)

    Apple, F. S.; Hellsten, Ylva; Clarkson, P. M.

    1988-01-01

    We could detect skeletal muscle injury early after an acute exercise bout by measuring creatine kinase (CK, EC 2.7.3.2) MM isoforms in serum. Eleven men performed 120 alternating-arm, eccentric (muscle lengthening) biceps contractions with the intensity of each contraction being 110% of maximal...... concentric strength--a form of exercise previously shown to cause significant increases of CK in serum at 24 h and muscle soreness 48 h after exercise. Total CK and CK-MM isoform activities in serum were determined before and at 0.5, 0.75, 1, 1.5, 2, and 6 h after exercise. Using thin-film agarose gels...... and a rapid isoelectric focusing technique, we separated the MM isoforms into MM3 (skeletal muscle form), MM2, and MM1 (in vivo conversion forms). The isoforms reflected the MM form released into the serum from tissue as well as the conversion of one form to another. There were no significant increases...

  13. Distinction between human cytochrome P450 (CYP) isoforms and identification of new phosphorylation sites by mass spectrometry

    DEFF Research Database (Denmark)

    Redlich, Gorden; Zanger, Ulrich M; Riedmaier, Stephan;

    2008-01-01

    In mammals, Cytochrome P450 (CYP) enzymes are bound to membranes of the endoplasmic reticulum and mitochondria, where they are responsible for the oxidative metabolism of many xenobiotics as well as organic endogenous compounds. In humans, 57 isoforms were identified which are classified based...

  14. INTERACTION OF PAH-RELATED COMPOUNDS WITH THE ALPHA AND BETA ISOFORMS OF ESTROGEN RECEPTOR. (R826192)

    Science.gov (United States)

    The ability of several 4- and 5-ring polycyclic aromatic hydrocarbons (PAHs), heterocyclic PAHs, and their monohydroxy derivatives to interact with the estrogen receptor (ER) alpha and beta isoforms was examined. Only compounds possessing a hydroxyl group were able to compete wit...

  15. Novel O-GlcNAcylation on Ser(40) of canonical H2A isoforms specific to viviparity.

    Science.gov (United States)

    Hirosawa, Mitsuko; Hayakawa, Koji; Yoneda, Chikako; Arai, Daisuke; Shiota, Hitoshi; Suzuki, Takehiro; Tanaka, Satoshi; Dohmae, Naoshi; Shiota, Kunio

    2016-01-01

    We report here newly discovered O-linked-N-acetylglucosamine (O-GlcNAc) modification of histone H2A at Ser(40) (H2AS40Gc). The mouse genome contains 18 H2A isoforms, of which 13 have Ser(40) and the other five have Ala(40). The combination of production of monoclonal antibody and mass spectrometric analyses with reverse-phase (RP)-high performance liquid chromatography (HPLC) fractionation indicated that the O-GlcNAcylation is specific to the Ser(40) isoforms. The H2AS40Gc site is in the L1 loop structure where two H2A molecules interact in the nucleosome. Targets of H2AS40Gc are distributed genome-wide and are dramatically changed during the process of differentiation in mouse trophoblast stem cells. In addition to the mouse, H2AS40Gc was also detected in humans, macaques and cows, whereas non-mammalian species possessing only the Ala(40) isoforms, such as silkworms, zebrafish and Xenopus showed no signal. Genome database surveys revealed that Ser(40) isoforms of H2A emerged in Marsupialia and persisted thereafter in mammals. We propose that the emergence of H2A Ser(40) and its O-GlcNAcylation linked a genetic event to genome-wide epigenetic events that correlate with the evolution of placental animals. PMID:27615797

  16. Myosin heavy-chain isoform distribution, fibre-type composition and fibre size in skeletal muscle of patients on haemodialysis

    DEFF Research Database (Denmark)

    Molsted, Stig; Eidemak, Inge; Sorensen, Helle Tauby;

    2007-01-01

    and distribution of muscle fibres and the myosin heavy-chain (MHC) isoform composition in patiens on haemodialysis (HD) in order to establish any differences with values for untrained control subjects. Material and methods. Muscle biopsies were obtained from the vastus lateralis muscle of 14 non-diabetic patients...

  17. Na(+)-K(+)-ATPase alpha(2)-isoform expression in guinea pig hearts during transition from compensation to decompensation.

    Science.gov (United States)

    Trouve, P; Carre, F; Belikova, I; Leclercq, C; Dakhli, T; Soufir, L; Coquard, I; Ramirez-Gil, J; Charlemagne, D

    2000-10-01

    Disturbance in ionic gradient across sarcolemma may lead to arrhythmias. Because Na(+)-K(+)-ATPase regulates intracellular Na(+) and K(+) concentrations, and therefore intracellular Ca(2+) concentration homeostasis, our aim was to determine whether changes in the Na(+)-K(+)-ATPase alpha-isoforms in guinea pigs during transition from compensated (CLVH) to decompensated left ventricular hypertrophy (DLVH) were concomitant with arrhythmias. After 12- and 20-mo aortic stenosis, CLVH and DLVH were characterized by increased mean arterial pressure (30% and 52.7%, respectively). DLVH differed from CLVH by significantly increased end-diastolic pressure (34%), decreased sarco(endo)plasmic reticulum Ca(2+)-ATPase (-75%), and increased Na(+)/Ca(2+) exchanger (25%) mRNA levels and by the occurrence of ventricular arrhythmias. The alpha-isoform (mRNA and protein levels) was significantly lower in DLVH (2.2 +/- 0.2- and 1. 4 +/- 0.15-fold, respectively, vs. control) than in CLVH (3.5 +/- 0. 4- and 2.2 +/- 0.13-fold, respectively) and was present in sarcolemma and T tubules. Changes in the levels of alpha(1)- and alpha(3)-isoform in CLVH and DLVH appear physiologically irrelevant. We suggest that the increased level of alpha(2)-isoform in CLVH may participate in compensation, whereas its relative decrease in DLVH may enhance decompensation and arrhythmias. PMID:11009487

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2016-09-15

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

  20. PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM IS IDENTIFIED AS THE 70-KILODALTON HEAT SHOCK PROTEIN HSPA2

    Science.gov (United States)

    THE PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM IS IDENTIFIED AS THE 70 kDa HEAT SHOCK PROTEIN HSPA2* Gabor Huszar1, Kathryn Stone2, David Dix3 and Lynne Vigue11The Sperm Physiology Laboratory, Department of Obstetrics and Gynecology, 2 W.M. Keck Foundatio...

  1. Two new isoforms of the human hepatoma-derived growth factor interact with components of the cytoskeleton.

    Science.gov (United States)

    Nüße, Jessica; Mirastschijski, Ursula; Waespy, Mario; Oetjen, Janina; Brandes, Nadine; Rebello, Osmond; Paroni, Federico; Kelm, Sørge; Dietz, Frank

    2016-05-01

    Hepatoma-derived growth factor (HDGF) is involved in diverse, apparently unrelated processes, such as cell proliferation, apoptosis, DNA-repair, transcriptional control, ribosome biogenesis and cell migration. Most of the interactions of HDGF with diverse molecules has been assigned to the hath region of HDGF. In this study we describe two previously unknown HDGF isoforms, HDGF-B and HDGF-C, generated via alternative splicing with structurally unrelated N-terminal regions of their hath region, which is clearly different from the well described isoform, HDGF-A. In silico modeling revealed striking differences near the PHWP motif, an essential part of the binding site for glycosaminoglycans and DNA/RNA. This observation prompted the hypothesis that these isoforms would have distinct interaction patterns with correspondingly diverse roles on cellular processes. Indeed, we discovered specific associations of HDGF-B and HDGF-C with cytoskeleton elements, such as tubulin and dynein, suggesting previously unknown functions of HDGF in retrograde transport, site directed localization and/or cytoskeleton organization. In contrast, the main isoform HDGF-A does not interact directly with the cytoskeleton, but via RNA with messenger ribonucleoprotein (mRNP) complexes. In summary, the discovery of HDGF splice variants with their discrete binding activities and subcellular distributions opened new avenues for understanding its biological function and importance. PMID:26845719

  2. Apolipoprotein E: isoform specific differences in tertiary structure and interaction with amyloid-β in human Alzheimer brain.

    Directory of Open Access Journals (Sweden)

    Phillip B Jones

    Full Text Available We applied a novel application of FLIM-FRET to in situ measurement and quantification of protein interactions to explore isoform specific differences in Aβ-ApoE interaction and ApoE tertiary conformation in senile plaques in human Alzheimer brain. ApoE3 interacts more closely with Aβ than ApoE4, but a greater proportion of Aβ molecules within plaques are decorated with ApoE4 than ApoE3, lending strong support to the hypothesis that isoform specific differences in ApoE are linked with Aβ deposition. We found an increased number of ApoE N-terminal fragments in ApoE4 plaques, consistent with the observation that ApoE4 is more easily cleaved than ApoE3. In addition, we measured a small but significant isoform specific difference in ApoE domain interaction. Based on our in situ data, supported by traditional biochemical data, we propose a pathway by which isoform specific conformational differences increase the level of cleavage at the hinge region of ApoE4, leading to a loss of ApoE function to mediate clearance of Aβ and thereby increase the risk of AD for carriers of the APOEε4 allele.

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

    Directory of Open Access Journals (Sweden)

    Nathalie Esber

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

  4. The three isoforms of the light-harvesting complex II Spectroscopic features, trimer formation, and functional roles

    CERN Document Server

    Standfuss, Jorg

    2004-01-01

    The major light-harvesting complex (LHC-II) of higher plants plays a crucial role in capturing light energy for photosynthesis and in regulating the flow of energy within the photosynthetic apparatus. Native LHC-II isolated from plant tissue consists of three isoforms, Lhcb1, Lhcb2, and Lhcb3, which form homo- and heterotrimers. All three isoforms are highly conserved among different species, suggesting distinct functional roles. We produced the three LHC-II isoforms by heterologous expression of the polypeptide in Escherichia coli and in vitro refolding with purified pigments. Although Lhcb1 and Lhcb2 are very similar in polypeptide sequence and pigment content, Lhcb3 is clearly different because it lacks an N-terminal phosphorylation site and has a higher chlorophyll a/b ratio, suggesting the absence of one chlorophyll b. Low temperature absorption and fluorescence emission spectra of the pure isoforms revealed small but significant differences in pigment organization. The oligomeric state of the pure isofo...

  5. Design, synthesis, and structure-activity analysis of isoform-selective retinoic acid receptor ß ligands

    DEFF Research Database (Denmark)

    Lund, Birgitte W.; Knapp, Anne Eeg; Piu, Fabrice;

    2009-01-01

    We recently discovered the isoform selective RAR beta 2 ligand 4'-octyl-4-biphenylcarboxylic acid (3, AC-55649). Although 3 is highly potent at RAR beta 2 and displays excellent selectivity, solubility issues make it unsuitable for drug development. Herein we describe the exploration of the SAR in...

  6. Different BAG-1 isoforms have distinct functions in modulating chemotherapeutic-induced apoptosis in breast cancer cells

    Institute of Scientific and Technical Information of China (English)

    Hongyu LIU; Zhuomin WANG; Yun BAI; Min WANG; Ying LI; Sen WEI; Qinghua ZHOU; Jun CHEN

    2009-01-01

    Aim:BAG-1 is a multifunctional anti-apoptotic gene with four isoforms,and different BAG-1 isoforms have different anti-apoptotic functions.In this study,we transfected BAG-1 isoforms into the human breast cancer cell lines Hs578T (ER nega-tive) and MCF-7 (ER positive) to study their effect on apoptosis with or without estrogens.Methods: The constructed recombinant expression vectors carrying individual BAG-1 isoforms was used to transfect human breast cancer cell lines Hs578T (ER negative) and MCF-7 (ER positive).After stable cell lines were made,a variety of apoptosis-inducing agents,including doxorubicin,docetaxel,and 5-FU,was used to treat these cell lines with or without estrogen to test the role of BAG-1.The mechanism by which BAG-1 affected the function of Bcl-2 was exploredby using the cycloheximide chase assay.Results: The BAG-1 p50 and p46 isoforms significantly enhanced the resistance to apoptosis in both cell lines according to flow cytometry analysis.BAG-1 p33 and p29 failed to protect the transfected cells from apoptosis.The cell viability assay showed that only BAG-1 p50,but not p46,p33,or p29,increased estrogen-dependent function in ER-positive cell line MCF-7.Only BAG-1 p50 dramatically increased its anti-apoptotic ability in the presence of estrogen,while estrogen has very little effect on the anti-apoptotic ability of other BAG-1 isoforms.In the detection of the expression of K-ras,Hsp70,cytochrome c,Raf-1,ER-α,and Bcl-2 in MCF-7 cells by Western blot,only Bcl-2 protein expression was significantly increased in MCF-7 cells transfected with BAG-1 p50 and p46,respectively.Furthermore,the cycloheximide chase assay indicated that the degradation of Bcl-2 protein was extended in the BAG-1 p50 and p46 transfected MCF-7 cells.Conclusion: Distinct isoforrns of BAG-1 have different anti-apoptotic functions in breast cancer cells,and that the BAG-1 p50 isoform can potentiate the role of estrogen in ER-positive breast cancer.

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

    Science.gov (United States)

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

    2010-01-01

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

  8. Charged residues distribution modulates selectivity of the open state of human isoforms of the voltage dependent anion-selective channel.

    Science.gov (United States)

    Amodeo, Giuseppe Federico; Scorciapino, Mariano Andrea; Messina, Angela; De Pinto, Vito; Ceccarelli, Matteo

    2014-01-01

    Voltage Dependent Anion-selective Channels (VDACs) are pore-forming proteins located in the outer mitochondrial membrane. They are responsible for the access of ions and energetic metabolites into the inner membrane transport systems. Three VDAC isoforms exist in mammalian, but their specific role is unknown. In this work we have performed extensive (overall ∼5 µs) Molecular Dynamics (MD) simulations of the human VDAC isoforms to detect structural and conformational variations among them, possibly related to specific functional roles of these proteins. Secondary structure analysis of the N-terminal domain shows a high similarity among the three human isoforms of VDAC but with a different plasticity. In particular, the N-terminal domain of the hVDAC1 is characterized by a higher plasticity, with a ∼20% occurrence for the 'unstructured' conformation throughout the folded segment, while hVDAC2, containing a peculiar extension of 11 amino acids at the N-terminal end, presents an additional 310-helical folded portion comprising residues 10' to 3, adhering to the barrel wall. The N-terminal sequences of hVDAC isoforms are predicted to have a low flexibility, with possible consequences in the dynamics of the human VDACs. Clear differences were found between hVDAC1 and hVDAC3 against hVDAC2: a significantly modified dynamics with possible important consequence on the voltage-gating mechanism. Charge distribution inside and at the mouth of the pore is responsible for a different preferential localization of ions with opposite charge and provide a valuable rationale for hVDAC1 and hVDAC3 having a Cl-/K+ selectivity ratio of 1.8, whereas hVDAC2 of 1.4. Our conclusion is that hVDAC isoforms, despite sharing a similar scaffold, have modified working features and a biological work is now requested to give evidence to the described dissimilarities. PMID:25084457

  9. Charged residues distribution modulates selectivity of the open state of human isoforms of the voltage dependent anion-selective channel.

    Directory of Open Access Journals (Sweden)

    Giuseppe Federico Amodeo

    Full Text Available Voltage Dependent Anion-selective Channels (VDACs are pore-forming proteins located in the outer mitochondrial membrane. They are responsible for the access of ions and energetic metabolites into the inner membrane transport systems. Three VDAC isoforms exist in mammalian, but their specific role is unknown. In this work we have performed extensive (overall ∼5 µs Molecular Dynamics (MD simulations of the human VDAC isoforms to detect structural and conformational variations among them, possibly related to specific functional roles of these proteins. Secondary structure analysis of the N-terminal domain shows a high similarity among the three human isoforms of VDAC but with a different plasticity. In particular, the N-terminal domain of the hVDAC1 is characterized by a higher plasticity, with a ∼20% occurrence for the 'unstructured' conformation throughout the folded segment, while hVDAC2, containing a peculiar extension of 11 amino acids at the N-terminal end, presents an additional 310-helical folded portion comprising residues 10' to 3, adhering to the barrel wall. The N-terminal sequences of hVDAC isoforms are predicted to have a low flexibility, with possible consequences in the dynamics of the human VDACs. Clear differences were found between hVDAC1 and hVDAC3 against hVDAC2: a significantly modified dynamics with possible important consequence on the voltage-gating mechanism. Charge distribution inside and at the mouth of the pore is responsible for a different preferential localization of ions with opposite charge and provide a valuable rationale for hVDAC1 and hVDAC3 having a Cl-/K+ selectivity ratio of 1.8, whereas hVDAC2 of 1.4. Our conclusion is that hVDAC isoforms, despite sharing a similar scaffold, have modified working features and a biological work is now requested to give evidence to the described dissimilarities.

  10. The Role of MAPT Haplotype H2 and Isoform 1N/4R in Parkinsonism of Older Adults.

    Directory of Open Access Journals (Sweden)

    Guilherme T Valenca

    Full Text Available Recently, we have shown that the Parkinson's disease (PD susceptibility locus MAPT (microtubule associated protein tau is associated with parkinsonism in older adults without a clinical diagnosis of PD. In this study, we investigated the relationship between parkinsonian signs and MAPT transcripts by assessing the effect of MAPT haplotypes on alternative splicing and expression levels of the most common isoforms in two prospective clinicopathologic studies of aging.using regression analysis, controlling for age, sex, study and neuropathology, we evaluated 976 subjects with clinical, genotyping and brain pathology data for haplotype analysis. For transcript analysis, we obtained MAPT gene and isoform-level expression from the dorsolateral prefrontal cortex for 505 of these subjects.The MAPT H2 haplotype was associated with lower total MAPT expression (p = 1.2x10-14 and global parkinsonism at both study entry (p = 0.001 and proximate to death (p = 0.050. Specifically, haplotype H2 was primarily associated with bradykinesia in both assessments (p<0.001 and p = 0.008. MAPT total expression was associated with age and decreases linearly with advancing age (p<0.001. Analysing MAPT alternative splicing, the expression of 1N/4R isoform was inversely associated with global parkinsonism (p = 0.008 and bradykinesia (p = 0.008. Diminished 1N/4R isoform expression was also associated with H2 (p = 0.001.Overall, our results suggest that age and H2 are associated with higher parkinsonism score and decreased total MAPT RNA expression. Additionally, we found that H2 and parkinsonism are associated with altered expression levels of specific isoforms. These findings may contribute to the understanding of the association between MAPT locus and parkinsonism in elderly subjects and in some extent to age-related neurodegenerative diseases.

  11. Progesterone receptor isoforms PRA and PRB differentially contribute to breast cancer cell migration through interaction with focal adhesion kinase complexes.

    Science.gov (United States)

    Bellance, Catherine; Khan, Junaid A; Meduri, Geri; Guiochon-Mantel, Anne; Lombès, Marc; Loosfelt, Hugues

    2013-05-01

    Progesterone receptor (PR) and progestins affect mammary tumorigenesis; however, the relative contributions of PR isoforms A and B (PRA and PRB, respectively) in cancer cell migration remains elusive. By using a bi-inducible MDA-MB-231 breast cancer cell line expressing PRA and/or PRB, we analyzed the effect of conditional PR isoform expression. Surprisingly, unliganded PRB but not PRA strongly enhanced cell migration as compared with PR(-) cells. 17,21-Dimethyl-19-norpregna-4,9-dien-3,20-dione (R5020) progestin limited this effect and was counteracted by the antagonist 11β-(4-dimethyl-amino)-phenyl-17β-hydroxy-17-(1-propynyl)-estra-4,9-dien-3-one (RU486). Of importance, PRA coexpression potentiated PRB-mediated migration, whereas PRA alone was ineffective. PR isoforms differentially regulated expressions of major players of cell migration, such as urokinase plasminogen activator (uPA), its inhibitor plasminogen activator inhibitor type 1, uPA receptor (uPAR), and β1-integrin, which affect focal adhesion kinase (FAK) signaling. Moreover, unliganded PRB but not PRA enhanced FAK Tyr397 phosphorylation and colocalized with activated FAK in cell protrusions. Because PRB, as well as PRA, coimmunoprecipitated with FAK, both isoforms can interact with FAK complexes, depending on their respective nucleocytoplasmic trafficking. In addition, FAK degradation was coupled to R5020-dependent turnovers of PRA and PRB. Such an effect of PRB/PRA expression on FAK signaling might thus affect adhesion/motility, underscoring the implication of PR isoforms in breast cancer invasiveness and metastatic evolution with underlying therapeutic outcomes.

  12. Characterization of polymorphisms and isoforms of the Clostridium perfringens phospholipase C gene (plc) reveals high genetic diversity.

    Science.gov (United States)

    Siqueira, Flávia F; Almeida, Marcelle O; Barroca, Tatiana M; Horta, Carolina C R; Carmo, Anderson O; Silva, Rodrigo O S; Pires, Prhiscylla S; Lobato, Francisco C F; Kalapothakis, Evanguedes

    2012-10-12

    Clostridium perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is encoded by the plc gene and has been implicated in several diseases; however, only a few studies have described polymorphisms in this gene. The aim of this study was to analyze polymorphisms in the Cp-PLC nucleotide and amino acid sequences obtained from isolates from different regions and to compare them to Clostridium phospholipase C sequences deposited in the NCBI database. Environmental samples (sediment, poultry feed, sawdust) and stool samples (from poultry, bovine, swine, horse, caprine, bird, dog, rabbit, toucan) were collected from healthy and sick animals. A total of 73 isolates were analyzed with the majority of samples belonging to the toxin type A subtype and possessing the gene encoding for the beta-2 toxin. Comparison of plc gene sequences from respective isolates revealed a high genetic diversity in the nucleotide sequences of mature Cp-PLC. Sequence comparisons identified 30 amino acid substitutions and 34 isoforms including some isoforms with substitutions in amino acids critical to toxin function. Comparison of sequences obtained in this study to Cp-PLC sequences obtained from the NCBI database resulted in the identification of 11 common haplotypes and 22 new isoforms. Phylogenetic analysis of phospholipase C sequences obtained from other Clostridium species identified relationships previously described. This report describes a broad characterization of the genetic diversity in the C. perfringens plc gene resulting in the identification of various isoforms. A better understanding of sequences encoding phospholipase C isoforms may reveal changes associated with protein function and C. perfringens virulence.

  13. Runx2-I isoform contributes to fetal bone formation even in the absence of specific N-terminal amino acids.

    Directory of Open Access Journals (Sweden)

    Hideaki Okura

    Full Text Available The Runt-related transcription factor 2 (Runx2 gene encodes the transcription factor Runx2, which is the master regulator of osteoblast development; insufficiency of this protein causes disorders of bone development such as cleidocranial dysplasia. Runx2 has two isoforms, Runx2-II and Runx2-I, and production of each isoform is controlled by a unique promoter: a distal promoter (P1 and a proximal promoter (P2, respectively. Although several studies have focused on differences and similarities between the two Runx2 isoforms, their individual roles in bone formation have not yet been determined conclusively, partly because a Runx2-I-targeted mouse model is not available. In this study, we established a novel Runx2-manipulated mouse model in which the first ATG of Runx2-I was replaced with TGA (a stop codon, and a neomycin-resistant gene (neo cassette was inserted at the first intron of Runx2-I. Homozygous Runx2-Ineo/neo mice showed severely reduced expression of Runx2-I, whereas Runx2-II expression was largely retained. Runx2-Ineo/neo mice showed neonatal lethality, and in these mice, intramembranous ossification was more severely defective than endochondral ossification, presumably because of the greater involvement of Runx2-I, compared with that of Runx2-II in intramembranous ossification. Interestingly, the depletion of neo rescued the above-described phenotypes, indicating that the isoform-specific N-terminal region of Runx2-I is not functionally essential for bone development. Taken together, our results provide a novel clue leading to a better understanding of the roles of Runx2 isoforms in osteoblast development.

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

    Directory of Open Access Journals (Sweden)

    Belinda Baquero-Pérez

    2015-11-01

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

  15. Proteomic analysis of glutathione S-transferase isoforms in mouse liver mitochondria

    Institute of Scientific and Technical Information of China (English)

    Hai-Dan Sun; Ya-Wei Ru; Dong-Juan Zhang; Song-Yue Yin; Liang Yin; Ying-Ying Xie; You-Fei Guan; Si-Qi Liu

    2012-01-01

    AIM:To survey glutathione (GSH) S-transferase (GST)isoforms in mitochondria and to reveal the isoforms' biological significance in diabetic mice.METHODS:The presence of GSTs in mouse liver mitochondria was systematically screened by two proteomic approaches,namely,GSH affinity chromatography/two dimensional electrophoresis (2DE/MALDI TOF/TOFMS) and SDS-PAGE/LC ESI MS/MS.The proteomic results were further confirmed by Western blotting using monoclonal antibodies against GSTs.To evaluate the liver mitochondrial GSTs quantitatively,calibration curves were generated by the loading amounts of individual recombinant GST protein vs the relative intensities elicited from the Western blotting.An extensive comparison of the liver mitochondrial GSTs was conducted between normal and db/db diabetic mice.Student's t test was adopted for the estimation of regression and significant difference.RESULTS:Using GSH affinity/2DF/MALDI TOF/TOF MS,three GSTs,namely,alpha3,mu1 and pi1,were identified; whereas five GSTs,alpha3,mu1,pi1,kappa1 and zeta1,were detected in mouse liver mitochondria using SDS-PAGE/LC ESI MS/MS,of these GSTs,GST kappa1 was reported as a specific mitochondrial GST.The R2 values of regression ranged between values of about 0.86 and 0.98,which were acceptable for the quantification.Based on the measurement of the GST abundances in liver mitochondria of normal and diabetic mice,the four GSTs,alpha3,kappa1,mu1 and zeta1,were found to be almost comparable between the two sets of animals,whereas,lower GST pi1 was detected in the diabetic mice compared with normal ones,the signal of Western blotting in control and db/ db diabetic mice liver mitochondria is 134.61 ± 53.84vs 99.74 ± 46.2,with P < 0.05.CONCLUSION:Our results indicate that GSTs exist widely in mitochondria and its abundances of mitochondrial GSTs might be tissue-dependent and disease-related.

  16. Evolution of a TRIM5-CypA splice isoform in old world monkeys.

    Directory of Open Access Journals (Sweden)

    Ruchi M Newman

    2008-02-01

    Full Text Available The TRIM family proteins share a conserved arrangement of three adjacent domains, an N-terminal RING domain, followed by one or two B-boxes and a coiled-coil, which constitutes the tripartite-motif for which the family is named. However, the C-termini of TRIM proteins vary, and include at least nine evolutionarily distinct, unrelated protein domains. Antiviral restriction factor TRIM5alpha has a C-terminal B30.2/SPRY domain, which is the major determinant of viral target specificity. Here, we describe the evolution of a cyclophilin-A encoding exon downstream of the TRIM5 locus of Asian macaques. Alternative splicing gives rise to chimeric transcripts encoding the TRIM motif fused to a C-terminal CypA domain (TRIM5-CypA. We detected TRIM5-CypA chimeric transcripts in primary lymphocytes from two macaque species. These were derived in part from a CypA pseudogene in the TRIM5 locus, which is distinct from the previously described CypA insertion in TRIM5 of owl monkeys. The CypA insertion is linked to a mutation in the 3' splice site upstream of exon 7, which may prevent or reduce expression of the alpha-isoform. All pig-tailed macaques (M. nemestrina screened were homozygous for the CypA insertion. In contrast, the CypA-containing allele was present in 17% (17/101 of rhesus macaques (M. mulatta. The block to HIV-1 infection in lymphocytes from animals bearing the TRIM5-CypA allele was weaker than that in cells from wild type animals. HIV-1 infectivity remained significantly lower than SIV infectivity, but was not rescued by treatment with cyclosporine A. Thus, unlike owl monkey TRIMCyp, expression of the macaque TRIM5-CypA isoform does not result in increased restriction of HIV-1. Despite its distinct evolutionary origin, Macaca TRIM5-CypA has a similar domain arrangement and shares approximately 80% amino-acid identity with the TRIMCyp protein of owl monkeys. The independent appearance of TRIM5-CypA chimeras in two primate lineages constitutes a

  17. Myosin isoform expression in the prehensile tails of didelphid marsupials: functional differences between arboreal and terrestrial opossums.

    Science.gov (United States)

    Rupert, J E; Schmidt, E Cordero; Moreira-Soto, A; Herrera, B Rodríguez; Vandeberg, J L; Butcher, M T

    2014-08-01

    Prehensile tails are defined as having the ability to grasp objects and are commonly used as a fifth appendage during arboreal locomotion. Despite the independent evolution of tail prehensility in numerous mammalian genera, data relating muscle structure, physiology, and function of prehensile tails are largely incomplete. Didelphid marsupials make an excellent model to relate myosin heavy chain (MHC) isoform fiber type with structure/function of caudal muscles, as all opossums have a prehensile tail and tail use varies between arboreal and terrestrial forms. Expanding on our previous work in the Virginia opossum, this study tests the hypothesis that arboreal and terrestrial opossums differentially express faster versus slower MHC isoforms, respectively. MHC isoform expression and percent fiber type distribution were determined in the flexor caudae longus (FCL) muscle of Caluromys