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Sample records for pr isoform expression

  1. Expression of Glycogen Phosphorylase Isoforms in Cultured Muscle from Patients with McArdle's Disease Carrying the p.R771PfsX33 PYGM Mutation

    PubMed Central

    García-Consuegra, Inés; Rubio, Juan C.; Orozco, Anna; Arenas, Joaquin; Martín, Miguel A.; Lucia, Alejandro; Gómez-Foix, Anna M.; Martí, Ramon; Andreu, Antoni L.

    2010-01-01

    Background Mutations in the PYGM gene encoding skeletal muscle glycogen phosphorylase (GP) cause a metabolic disorder known as McArdle's disease. Previous studies in muscle biopsies and cultured muscle cells from McArdle patients have shown that PYGM mutations abolish GP activity in skeletal muscle, but that the enzyme activity reappears when muscle cells are in culture. The identification of the GP isoenzyme that accounts for this activity remains controversial. Methodology/Principal Findings In this study we present two related patients harbouring a novel PYGM mutation, p.R771PfsX33. In the patients' skeletal muscle biopsies, PYGM mRNA levels were ∼60% lower than those observed in two matched healthy controls; biochemical analysis of a patient muscle biopsy resulted in undetectable GP protein and GP activity. A strong reduction of the PYGM mRNA was observed in cultured muscle cells from patients and controls, as compared to the levels observed in muscle tissue. In cultured cells, PYGM mRNA levels were negligible regardless of the differentiation stage. After a 12 day period of differentiation similar expression of the brain and liver isoforms were observed at the mRNA level in cells from patients and controls. Total GP activity (measured with AMP) was not different either; however, the active GP activity and immunoreactive GP protein levels were lower in patients' cell cultures. GP immunoreactivity was mainly due to brain and liver GP but muscle GP seemed to be responsible for the differences. Conclusions/Significance These results indicate that in both patients' and controls' cell cultures, unlike in skeletal muscle tissue, most of the protein and GP activities result from the expression of brain GP and liver GP genes, although there is still some activity resulting from the expression of the muscle GP gene. More research is necessary to clarify the differential mechanisms of metabolic adaptations that McArdle cultures undergo in vitro. PMID:20957198

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

    PubMed Central

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

    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 isoforms, 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. PMID:18837087

  3. PKC Isoform Expression in Modeled Microgravity

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

  4. Promoter hypermethylation of progesterone receptor isoform B (PR-B) in endometriosis.

    PubMed

    Wu, Yan; Strawn, Estil; Basir, Zainab; Halverson, Gloria; Guo, Sun-Wei

    2006-01-01

    The physiological effects of progesterone (P) are mediated by two isoforms of progesterone receptors (PRs): PR-A and PR-B. Progestins have long been used in the treatment of endometriosis but unfortunately the relief of pain is relatively short-term. In addition, about nine percent of women with endometriosis simply do not respond to progestin therapy due to unknown reasons. In fact, a general tendency for relative progesterone resistance within eutopic and ectopic endometrium of women with endometriosis and also the downregulation of PR-B, but not PR-A, in endometriosis have been noted. Since promoter hypermethylation is well-documented to be associated with transcriptional silencing, we sought to determine the methylation status of the PR-A and PR-B promoter regions in the epithelial component of endometriotic implants using a combination of laser capture microdissection (LCM), methylation specific PCR, and bisulfite sequencing. We found that the promoter region of PR-B, but not PR-A, is hypermethylated in endometriosis as compared with controls. In addition, the PR-B expression was significantly reduced in the ectopic endometrium. Our finding suggests that progesterone resistance in endometriosis in general and the down regulation of PR-B, but not PR-A, in particular, are a result of promoter hypermethylation of PR-B, but not PR-A. This, in conjunction with our reported aberrant methylation of HOXA10 in the eutopic endometrium of women with endometriosis, strongly suggests that endometriosis is an epigenetic disease. This perspective should potentially open up new avenues for the delineation of pathogenesis of endometriosis, and might also lead to novel ways to treat the disease through reversing aberrant methylation via pharmacological means.

  5. Altered expression of progesterone receptor isoforms A and B in human eutopic endometrium in endometriosis patients.

    PubMed

    Wölfler, Monika Martina; Küppers, Mareike; Rath, Werner; Buck, Volker Uwe; Meinhold-Heerlein, Ivo; Classen-Linke, Irmgard

    2016-07-01

    Recent data implicate an altered expression of progesterone receptor isoform A (PR-A) and B (PR-B) in the endometrium of endometriosis patients. This prospective exploratory study aimed to precisely determine the PR-A and PR-B expression using immunohistochemical techniques in eutopic endometrium of women with endometriosis compared with disease-free women throughout the menstrual cycle. All symptomatic patients underwent laparoscopy for the diagnosis of endometriosis and histological confirmation of the disease (EO) whereas controls were proven disease-free (CO). In CO samples (n=10) an increased expression of PR-A and PR-B during the proliferative to early secretory phase and a decreased expression of both receptor isoforms during the mid to late secretory phase was ascertained in accordance with previous studies. In patients with endometriosis (n=16) no cycle dependent pattern of PR-A and PR-B expression was identified in contrast to patients without endometriosis. Moreover, in EO samples a huge variety of inter- and intra-individual differences in PR-A and PR-B expression were detected. These data provide further evidence that dysregulation of the PR-A and PR-B expression might contribute to the pathophysiology of endometriosis.

  6. Expression of Contractile Protein Isoforms in Microgravity

    NASA Technical Reports Server (NTRS)

    Anderson, Page A. W.

    1996-01-01

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

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

    PubMed

    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.

  8. Regulation of PGC-1α Isoform Expression in Skeletal Muscles

    PubMed Central

    Popov, D. V.; Lysenko, E. A.; Kuzmin, I. V.; Vinogradova, Vinogradova; Grigoriev, A. I.

    2015-01-01

    The coactivator PGC-1α is the key regulator of mitochondrial biogenesis in skeletal muscle. Skeletal muscle expresses several PGC-1α isoforms. This review covers the functional role of PGC-1α isoforms and the regulation of their exercise-associated expression in skeletal muscle. The patterns of PGC-1α mRNA expression may markedly differ at rest and after muscle activity. Different signaling pathways are activated by different physiological stimuli, which regulate the expression of the PGC-1α gene from the canonical and alternative promoters: expression from a canonical (proximal) promoter is regulated by activation of the AMPK; expression from an alternative promoter, via a β2-adrenergic receptor. All transcripts from both promoters are subject to alternative splicing. As a result, truncated isoforms that possess different properties are translated: truncated isoforms are more stable and predominantly activate angiogenesis, whereas full-length isoforms manly regulate mitochondrial biogenesis. The existence of several isoforms partially explains the broad-spectrum function of this protein and allows the organism to adapt to different physiological stimuli. Regulation of the PGC-1α gene expression by different signaling pathways provides ample opportunity for pharmacological influence on the expression of this gene. Those opportunities might be important for the treatment and prevention of various diseases, such as metabolic syndrome and diabetes mellitus. Elucidation of the regulatory mechanisms of the PGC-1α gene expression and their functional role may provide an opportunity to control the expression of different isoforms through exercise and/or pharmacological intervention. PMID:25927001

  9. Prion propagation in cells expressing PrP glycosylation mutants.

    PubMed

    Salamat, Muhammad K; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

    2011-04-01

    Infection by prions involves conversion of a host-encoded cell surface protein (PrP(C)) to a disease-related isoform (PrP(Sc)). PrP(C) carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrP(C) glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrP(Sc) and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrP(Sc), while PrP(C) with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrP(C), were able to form infectious PrP(Sc). Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection.

  10. Mast cells express novel functional IL-15 receptor alpha isoforms.

    PubMed

    Bulanova, Elena; Budagian, Vadim; Orinska, Zane; Krause, Hans; Paus, Ralf; Bulfone-Paus, Silvia

    2003-05-15

    Mast cells previously have been reported to be regulated by IL-15 and to express a distinct IL-15R, termed IL-15RX. To further examine IL-15 binding and signaling in mast cells, we have studied the nature of the IL-15R and some of its biological activities in these cells. In this study, we report the existence of three novel isoforms of the IL-15R alpha chain in murine bone marrow-derived mast cells as a result of an alternative exon-splicing mechanism within the IL-15R alpha gene. These correspond to new mRNA transcripts lacking exon 4; exons 3 and 4; or exons 3, 4, and 5 (IL-15R alpha Delta 4, IL-15R alpha Delta 3,4, IL-15R alpha Delta 3,4,5). After transient transfection in COS-7 cells, all IL-15R alpha isoforms associate with the Golgi apparatus, the endoplasmic reticulum, the perinuclear space, and the cell membrane. Analysis of glycosylation pattern demonstrates the usage of a single N-glycosylation site, while no O-glycosylation is observed. Importantly, IL-15 binds with high affinity to, and promotes the survival of, murine BA/F3 cells stably transfected with the IL-15R alpha isoforms. Furthermore, we report that signaling mediated by IL-15 binding to the newly identified IL-15R alpha isoforms involves the phosphorylation of STAT3, STAT5, STAT6, Janus kinase 2, and Syk kinase. Taken together, our data indicate that murine mast cells express novel, fully functional IL-15R alpha isoforms, which can explain the selective regulatory effects of IL-15 on these cells.

  11. Transgenetic studies implicate interactions between homologous PrP isoforms in scrapie prion replication.

    PubMed

    Prusiner, S B; Scott, M; Foster, D; Pan, K M; Groth, D; Mirenda, C; Torchia, M; Yang, S L; Serban, D; Carlson, G A

    1990-11-16

    Transgenic (Tg) mice expressing both Syrian hamster (Ha) and mouse (Mo) prion protein (PrP) genes were used to probe the mechanism of scrapie prion replication. Four Tg lines expressing HaPrP exhibited distinct incubation times ranging from 48 to 277 days, which correlated inversely with HaPrP mRNA and HaPrPC. Bioassays of Tg brain extracts showed that the prion inoculum dictates which prions are synthesized de novo. Tg mice inoculated with Ha prions had approximately 10(9) ID50 units of Ha prions per gram of brain and less than 10 units of Mo prions. Conversely, Tg mice inoculated with Mo prions synthesized Mo prions but not Ha prions. Similarly, Tg mice inoculated with Ha prions exhibited neuropathologic changes characteristic of hamsters with scrapie, while Mo prions produced changes similar to those in non-Tg mice. Our results argue that species specificity of scrapie prions resides in the PrP sequence and prion synthesis is initiated by a species-specific interaction between PrPSc in the inoculum and homologous PrPC.

  12. Differential expression of two scribble isoforms during Drosophila embryogenesis.

    PubMed

    Li, M; Marhold, J; Gatos, A; Török, I; Mechler, B M

    2001-10-01

    The tumour suppressor gene scribble (scrib) is required for epithelial polarity and growth control in Drosophila. Here, we report the identification and embryonic expression pattern of two Scrib protein isoforms resulting from alternative splicing during scrib transcription. Both proteins are first ubiquitously expressed during early embryogenesis. Then, during morphogenesis each Scrib protein displays a specific pattern of expression in the central and peripheral nervous systems, CNS and PNS, respectively. During germ band extension, the expression of the longer form Scrib1 occurs predominantly in the neuroblasts derived from the neuro-ectoderm and becomes later restricted to CNS neurones as well as to the pole cells in the gonads. By contrast, the shorter form Scrib2 is strongly expressed in the PNS and a subset of CNS neurones.

  13. Quantification of spatiotemporal patterns of Ras isoform expression during development

    PubMed Central

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

    2017-01-01

    Ras proteins are important signalling hubs frequently dysregulated in cancer and in a group of developmental disorders called Rasopathies. Three Ras genes encode four proteins that differentially contribute to these phenotypes. Using quantitative real-time PCR (qRT-PCR) we have measured the gene expression profiles of each of the Ras isoforms in a panel of mouse tissues derived from a full developmental time course spanning embryogenesis through to adulthood. In most tissues and developmental stages we observe a relative contribution of KRas4B > > NRas ≥ KRas4A > HRas to total Ras expression with KRas4B typically representing 60–99% of all Ras transcripts. KRas4A is the most dynamically regulated Ras isoform with significant up-regulation of expression observed pre-term in stomach, intestine, kidney and heart. The expression patterns assist interpretation of the essential role of KRas in development and the preponderance of KRas mutations in cancer. PMID:28117393

  14. Dienogest, a synthetic progestin, down-regulates expression of CYP19A1 and inflammatory and neuroangiogenesis factors through progesterone receptor isoforms A and B in endometriotic cells.

    PubMed

    Ichioka, Masayuki; Mita, Shizuka; Shimizu, Yutaka; Imada, Kazunori; Kiyono, Tohru; Bono, Yukiko; Kyo, Satoru

    2015-03-01

    Dienogest (DNG) is a selective progesterone receptor (PR) agonist and oral administration of DNG is used for the treatment of endometriosis. DNG is considered to act on PR to down-regulate pathophysiological factors associated with endometriosis. PR exists as two major isoforms, PR-A and PR-B, and their physiological functions are mostly distinct. It was suggested that PR isoform expression patterns are altered in endometriosis, but it is unknown whether the pharmacological effects of DNG are exerted through PR-A, PR-B or both. In the present study, we investigated the pharmacological effects of DNG through these PR isoforms on the expression of CYP19A1 which encodes aromatase and inflammatory and neuroangiogenesis factors associated with the pain and progression of endometriosis. We used immortalized human endometriotic epithelial cell lines that specifically express PR-A or PR-B in a spheroid cell culture system, and treated them with DNG. We evaluated messenger RNA (mRNA) expression of CYP19A1, prostaglandin (PG)E2 synthase (cyclooxygenase (COX)-2 and microsomal PGE2 synthase (mPGES)-1), inflammatory cytokines (interleukin (IL)-6, IL-8, and monocyte chemoattractant protein (MCP)-1) and neuroangiogenesis factors (vascular endothelial growth factor (VEGF) and nerve growth factor (NGF)) using real-time polymerase chain reaction. In addition, PGE2 production was measured by enzyme immunoassay. We found that DNG down-regulated mRNA expression of CYP19A1, COX-2, mPGES-1, IL-6, IL-8, MCP-1, NGF and VEGF, and PGE2 production in human endometriotic epithelial cell lines that specifically express either PR-A or PR-B. These results demonstrate that DNG activates both PR-A and PR-B and down-regulates the expression of pathophysiological factors associated with pain and progression of endometriosis. Our results suggest that DNG exerts therapeutic efficacy against the pain and progression of endometriosis regardless of PR isoform expression patterns.

  15. Expression of Phosphoinositide-Specific Phospholipase C Isoforms in Native Endothelial Cells

    PubMed Central

    Béziau, Delphine M.; Toussaint, Fanny; Blanchette, Alexandre; Dayeh, Nour R.; Charbel, Chimène; Tardif, Jean-Claude; Dupuis, Jocelyn; Ledoux, Jonathan

    2015-01-01

    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 in the

  16. Expression of CD44 isoforms in renal cell tumors. Positive correlation to tumor differentiation.

    PubMed Central

    Terpe, H. J.; Störkel, S.; Zimmer, U.; Anquez, V.; Fischer, C.; Pantel, K.; Günthert, U.

    1996-01-01

    CD44 isoforms have been implicated in tumor progression and embryogenesis. Primary renal cell tumors (n = 100) of various histopathological differentiation and grading stages were analyzed for expression of CD44 isoforms in comparison with nonmalignant adult and fetal renal tissues. Evaluations were performed by immunohistochemistry using CD44 isoform-specific monoclonal antibodies and by reverse transcriptase polymerase chain reactions (RT-PCR). In the nonmalignant kidney no CD44 variant isoforms were detected. There was a significant increase in expression of CD44 standard (CD44s) and several variant isoforms (CD44v) in the course of tumor differentiation in clear cell carcinomas (n = 68) from stages G1 to G3 (P < 0.0001 for CD44s and isoforms containing CD44-6v, and P < 0.007 for those containing CD44-9v). Also, in chromophilic cell carcinomas (n = 13), CD44 isoform expression correlated with grading; ie, no CD44 expression was detected in G1 tumors, whereas in approximately 50% of the G2 tumors, CD44s, CD44-6v, and CD44-9v isoforms were present. Oncocytomas (n = 8), which are benign renal cell tumors, did not express CD44 isoforms, whereas invasive chromophobe cell carcinomas (n = 11) were positive for CD44s and CD44v isoforms. Transcript analyses by RT-PCR revealed that the upregulated isoforms in the carcinoma cells contained exons 8 to 10 and 3, 8 to 10 in combination from the variant region. In conclusion, expression of variant CD44 isoforms was strongly correlated with grading and appears to mediate a more aggressive phenotype to renal cell tumors. Images Figure 3 Figure 4 Figure 5 Figure 6 PMID:8579108

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

    PubMed

    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.

  18. Robust stratification of breast cancer subtypes using differential patterns of transcript isoform expression.

    PubMed

    Stricker, Thomas P; Brown, Christopher D; Bandlamudi, Chaitanya; McNerney, Megan; Kittler, Ralf; Montoya, Vanessa; Peterson, April; Grossman, Robert; White, Kevin P

    2017-03-01

    Breast cancer, the second leading cause of cancer death of women worldwide, is a heterogenous disease with multiple different subtypes. These subtypes carry important implications for prognosis and therapy. Interestingly, it is known that these different subtypes not only have different biological behaviors, but also have distinct gene expression profiles. However, it has not been rigorously explored whether particular transcriptional isoforms are also differentially expressed among breast cancer subtypes, or whether transcript isoforms from the same sets of genes can be used to differentiate subtypes. To address these questions, we analyzed the patterns of transcript isoform expression using a small set of RNA-sequencing data for eleven Estrogen Receptor positive (ER+) subtype and fourteen triple negative (TN) subtype tumors. We identified specific sets of isoforms that distinguish these tumor subtypes with higher fidelity than standard mRNA expression profiles. We found that alternate promoter usage, alternative splicing, and alternate 3'UTR usage are differentially regulated in breast cancer subtypes. Profiling of isoform expression in a second, independent cohort of 68 tumors confirmed that expression of splice isoforms differentiates breast cancer subtypes. Furthermore, analysis of RNAseq data from 594 cases from the TCGA cohort confirmed the ability of isoform usage to distinguish breast cancer subtypes. Also using our expression data, we identified several RNA processing factors that were differentially expressed between tumor subtypes and/or regulated by estrogen receptor, including YBX1, YBX2, MAGOH, MAGOHB, and PCBP2. RNAi knock-down of these RNA processing factors in MCF7 cells altered isoform expression. These results indicate that global dysregulation of splicing in breast cancer occurs in a subtype-specific and reproducible manner and is driven by specific differentially expressed RNA processing factors.

  19. Robust stratification of breast cancer subtypes using differential patterns of transcript isoform expression

    PubMed Central

    Stricker, Thomas P.; Bandlamudi, Chaitanya; Kittler, Ralf; Montoya, Vanessa; Peterson, April; Grossman, Robert

    2017-01-01

    Breast cancer, the second leading cause of cancer death of women worldwide, is a heterogenous disease with multiple different subtypes. These subtypes carry important implications for prognosis and therapy. Interestingly, it is known that these different subtypes not only have different biological behaviors, but also have distinct gene expression profiles. However, it has not been rigorously explored whether particular transcriptional isoforms are also differentially expressed among breast cancer subtypes, or whether transcript isoforms from the same sets of genes can be used to differentiate subtypes. To address these questions, we analyzed the patterns of transcript isoform expression using a small set of RNA-sequencing data for eleven Estrogen Receptor positive (ER+) subtype and fourteen triple negative (TN) subtype tumors. We identified specific sets of isoforms that distinguish these tumor subtypes with higher fidelity than standard mRNA expression profiles. We found that alternate promoter usage, alternative splicing, and alternate 3’UTR usage are differentially regulated in breast cancer subtypes. Profiling of isoform expression in a second, independent cohort of 68 tumors confirmed that expression of splice isoforms differentiates breast cancer subtypes. Furthermore, analysis of RNAseq data from 594 cases from the TCGA cohort confirmed the ability of isoform usage to distinguish breast cancer subtypes. Also using our expression data, we identified several RNA processing factors that were differentially expressed between tumor subtypes and/or regulated by estrogen receptor, including YBX1, YBX2, MAGOH, MAGOHB, and PCBP2. RNAi knock-down of these RNA processing factors in MCF7 cells altered isoform expression. These results indicate that global dysregulation of splicing in breast cancer occurs in a subtype-specific and reproducible manner and is driven by specific differentially expressed RNA processing factors. PMID:28263985

  20. Differential expression of serum clusterin isoforms in colorectal cancer.

    PubMed

    Rodríguez-Piñeiro, Ana M; de la Cadena, María Páez; López-Saco, Angel; Rodríguez-Berrocal, Francisco J

    2006-09-01

    Clusterin is an enigmatic protein altered in tumors of colorectal cancer patients. Because there is no information available about serum clusterin regarding this pathology, we applied proteomic techniques to analyze its isoforms in donors and patients. First we separated serum proteins through concanavalin A, obtaining a fraction with non- and O-glycosylated proteins (FI) and a second fraction enriched in N-glycoproteins (FII) wherein clusterin was supposed to elute on the basis of its glycosylation. Surprisingly analysis of the FI fraction revealed the existence of an unexpected and aberrantly N-glycosylated clusterin that was overexpressed in patients and comprised at least five isoforms with different isoelectric points. On the other hand, two-dimensional electrophoretic analysis of the clusterin eluted in FII detected one isoform that was increased and 15 isoforms that were decreased or absent in serum of patients. Finally immunoquantification by slot blot showed that in total serum and in FI the clusterin levels were significantly increased in patients, whereas in FII there was no significant variation. Therefore, serum clusterin and some of its isoforms could have a potential value as colorectal tumor markers and are interesting subjects for biomarker studies.

  1. Regulated Expression of a Calmodulin Isoform Alters Growth and Development in Potato

    NASA Technical Reports Server (NTRS)

    Poovaiah, B. W.; Takezawa, D.; An, G.; Han, T.-J.

    1996-01-01

    A transgene approach was taken to study the consequences of altered expression of a calmodutin iso-form on plant growth and development. Eight genomic clones of potato calmodulin (PCM 1 to 8) have been isolated and characterized. Among the potato calmodulin isoforms studied, PCM 1 differs from the other isoforms because of its unique amino acid substitutions. Transgenic potato plants were produced carrying sense construct of PCM 1 fused to the CAMV 35S promoter. Transgenic plants showing a moderate increase in PCM 1 MRNA exhibited strong apical dominance, produced elongated tubers, and were taller than the controls. Interestingly, the plants expressing the highest level of PCM 1 MRNA did not form underground tubers. Instead, these transgenic plants produced aerial tubers when allowed to grow for longer periods. The expression of different calmodulin isoforms (PCM 1, 5, 6, and 8) was studied in transgenic plants. Among the four potato calmodulin isoforms, only the expression of PCM 1 MRNA was altered in transgenic plants, while the expression of other isoforms was not significantly altered. Western analysis revealed increased PCM 1 protein in transgenic plants, indicating that the expression of both MRNA and protein are altered in transgenic plants. These results suggest that increasing the expression of PCM 1 alters growth and development in potato plants.

  2. Enhanced expression of two discrete isoforms of matrix metalloproteinase-2 in experimental and human diabetic nephropathy

    PubMed Central

    Bae, Sun Sik; Lee, Min Young; Rhee, Harin; Kim, Il Young; Seong, Eun Young; Lee, Dong Won; Lee, Soo Bong; Kwak, Ihm Soo; Lovett, David H.

    2017-01-01

    Background We recently reported on the enhanced expression of two isoforms of matrix metalloproteinase-2 (MMP-2) in human renal transplantation delayed graft function. These consist of the conventional secreted, full length MMP-2 isoform (FL-MMP-2) and a novel intracellular N-Terminal Truncated isoform (NTT-MMP-2) generated by oxidative stress-mediated activation of an alternate promoter in the MMP-2 first intron. Here we evaluated the effect of hyperglycemia and diabetes mellitus on the in vitro and in vivo expression of the two MMP-2 isoforms. Methods We quantified the abundance of the FL-MMP-2 and NTT-MMP-2 transcripts by qPCR in HK2 cells cultured in high glucose or 4-hydroxy-2-hexenal (HHE) and tested the effects of the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC). The streptozotocin (STZ) murine model of Type I diabetes mellitus and renal biopsies of human diabetic nephropathy were used in this study. Results Both isoforms of MMP-2 in HK2 cells were upregulated by culture in high glucose or with HHE. PDTC treatment did not suppress high glucose-mediated FL-MMP-2 expression but potently inhibited NTT-MMP-2 expression. With STZ-treated mice, renal cortical expression of both isoforms was increased (FL-MMP-2, 1.8-fold; NTT-MMP-2, greater than 7-fold). Isoform-specific immunohistochemical staining revealed low, but detectable levels of the FL-MMP-2 isoform in controls, while NTT-MMP-2 was not detected. While there was a modest increase in tubular epithelial cell staining for FL-MMP-2 in STZ-treated mice, NTT-MMP-2 was intensely expressed in a basolateral pattern. FL-MMP-2 and NTT-MMP-2 isoform expression as quantified by qPCR were both significantly elevated in renal biopsies of human diabetic nephropathy (12-fold and 3-fold, respectively). Conclusions The expression of both isoforms of MMP-2 was enhanced in an experimental model of diabetic nephropathy and in human diabetic nephropathy. Selective MMP-2 isoform inhibition could offer a novel approach for

  3. Cell-Specific Expression of Plasma Membrane Calcium ATPase Isoforms in Retinal Neurons

    PubMed Central

    Krizaj, David; Demarco, Steven J.; Johnson, Juliette; Strehler, Emanuel E.; Copenhagen, David R.

    2007-01-01

    Ca2+ extrusion by high-affinity plasma membrane calcium ATPases (PMCAs) is a principal mechanism for the clearance of Ca2+ from the cytosol. The PMCA family consists of four isoforms (PMCA1–4). Little is known about the selective expression of these isoforms in brain tissues or about the physiological function conferred upon neurons by any given isoform. We investigated the cellular and subcellular distribution of PMCA isoforms in a mammalian retina. Mouse photoreceptors, cone bipolar cells and horizontal cells, which respond to light with a graded polarization, express isoform 1 (PMCA1) of the PMCA family. PMCA2 is localized to rod bipolar cells, horizontal cells, amacrine cells, and ganglion cells, and PMCA3 is predominantly expressed in spiking neurons, including both amacrine and ganglion cells but is also found in horizontal cells. PMCA4 was found to be selectively expressed in both synaptic layers. Optical measurements of Ca2+ clearance showed that PMCAs mediate Ca2+ extrusion in both rod and cone bipolar cells. In addition, we found that rod bipolar cells, but not cone bipolar cells possess a prominent Na+/Ca2+ exchange mechanism. We conclude that PMCA isoforms are selectively expressed in retinal neurons and that processes of Ca2+ clearance are different in rod and cone bipolar cells. PMID:12209837

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

    PubMed Central

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

    2015-01-01

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

  5. Transgenic Rabbits Expressing Ovine PrP Are Susceptible to Scrapie.

    PubMed

    Sarradin, Pierre; Viglietta, Céline; Limouzin, Claude; Andréoletti, Olivier; Daniel-Carlier, Nathalie; Barc, Céline; Leroux-Coyau, Mathieu; Berthon, Patricia; Chapuis, Jérôme; Rossignol, Christelle; Gatti, Jean-Luc; Belghazi, Maya; Labas, Valérie; Vilotte, Jean-Luc; Béringue, Vincent; Lantier, Frédéric; Laude, Hubert; Houdebine, Louis-Marie

    2015-08-01

    Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases affecting a wide range of mammalian species. They are caused by prions, a proteinaceous pathogen essentially composed of PrPSc, an abnormal isoform of the host encoded cellular prion protein PrPC. Constrained steric interactions between PrPSc and PrPC are thought to provide prions with species specificity, and to control cross-species transmission into other host populations, including humans. Transgenetic expression of foreign PrP genes has been successfully and widely used to overcome the recognized resistance of mouse to foreign TSE sources. Rabbit is one of the species that exhibit a pronounced resistance to TSEs. Most attempts to infect experimentally rabbit have failed, except after inoculation with cell-free generated rabbit prions. To gain insights on the molecular determinants of the relative resistance of rabbits to prions, we generated transgenic rabbits expressing the susceptible V136R154Q171 allele of the ovine PRNP gene on a rabbit wild type PRNP New Zealand background and assessed their experimental susceptibility to scrapie prions. All transgenic animals developed a typical TSE 6-8 months after intracerebral inoculation, whereas wild type rabbits remained healthy more than 700 days after inoculation. Despite the endogenous presence of rabbit PrPC, only ovine PrPSc was detectable in the brains of diseased animals. Collectively these data indicate that the low susceptibility of rabbits to prion infection is not enciphered within their non-PrP genetic background.

  6. Influence of development on Na(+)/K(+)-ATPase expression: isoform- and tissue-dependency.

    PubMed

    Lopez, Luciane B; Quintas, Luis Eduardo M; Noël, François

    2002-02-01

    The four isoforms of the catalytic subunit of Na(+)/K(+)-ATPase identified in rats differ in their affinities for ions and ouabain. Moreover, its expression is tissue-specific, developmentally and hormonally regulated. The aim of the present work was to evaluate the influence of age on the ratio and density of these isoforms in crude membrane preparations from rat brain hemispheres, brainstem, heart ventricles and kidneys. In all tissues investigated, Na(+)/K(+)-ATPase activity was higher in adults than in neonates but brain tissues presented the most remarkable differences. In these tissues, ouabain inhibition curves for Na(+)/K(+)-ATPase activity revealed the presence of two processes with different sensitivities to ouabain. An increase of approximately sixfold in the expression of the high affinity isoforms was observed between newborn and adult rats. In contrast, the low affinity isoform increased only approximately twofold in brainstem whereas it increased ninefold in brain hemispheres. Unlike brain tissues, a decrease (almost fourfold) in the number of high affinity ouabain binding sites was observed during ontogenesis of the heart. Although limited by the inability to resolve alpha(2) and alpha(3) isoforms, present data indicate that the influence of development on the expression of Na(+)/K(+)-ATPase depends not only on the isoform, but also on the tissue where the enzyme is expressed.

  7. Nutritional control of mRNA isoform expression during developmental arrest and recovery in C. elegans.

    PubMed

    Maxwell, Colin S; Antoshechkin, Igor; Kurhanewicz, Nicole; Belsky, Jason A; Baugh, L Ryan

    2012-10-01

    Nutrient availability profoundly influences gene expression. Many animal genes encode multiple transcript isoforms, yet the effect of nutrient availability on transcript isoform expression has not been studied in genome-wide fashion. When Caenorhabditis elegans larvae hatch without food, they arrest development in the first larval stage (L1 arrest). Starved larvae can survive L1 arrest for weeks, but growth and post-embryonic development are rapidly initiated in response to feeding. We used RNA-seq to characterize the transcriptome during L1 arrest and over time after feeding. Twenty-seven percent of detectable protein-coding genes were differentially expressed during recovery from L1 arrest, with the majority of changes initiating within the first hour, demonstrating widespread, acute effects of nutrient availability on gene expression. We used two independent approaches to track expression of individual exons and mRNA isoforms, and we connected changes in expression to functional consequences by mining a variety of databases. These two approaches identified an overlapping set of genes with alternative isoform expression, and they converged on common functional patterns. Genes affecting mRNA splicing and translation are regulated by alternative isoform expression, revealing post-transcriptional consequences of nutrient availability on gene regulation. We also found that phosphorylation sites are often alternatively expressed, revealing a common mode by which alternative isoform expression modifies protein function and signal transduction. Our results detail rich changes in C. elegans gene expression as larvae initiate growth and post-embryonic development, and they provide an excellent resource for ongoing investigation of transcriptional regulation and developmental physiology.

  8. Differential expression of IGF-1 mRNA isoforms in colorectal carcinoma and normal colon tissue.

    PubMed

    Kasprzak, Aldona; Szaflarski, Witold; Szmeja, Jacek; Andrzejewska, Małgorzata; Przybyszewska, Wiesława; Kaczmarek, Elżbieta; Koczorowska, Maria; Kościński, Tomasz; Zabel, Maciej; Drews, Michał

    2013-01-01

    The insulin-like growth factor (IGF)-1 gene consists of 6 exons resulting in the expression of 6 variant forms of mRNA (IA, IB, IC, IIA, IIB and IIC) due to an alternative splicing. The mechanisms of IGF-1 gene splicing and the role of local expression manifested by IGF-1 mRNA variants in colorectal carcinoma (CRC) have not been extensively investigated. Therefore, the aim of our study was to analyse the expression of IGF-1 mRNA isoforms [A, B, C, P1 (class I) and P2 (class II)], as well as the protein expression in CRC and control samples isolated from 28 patients. The expression of Ki-67 was also analysed and clinical data were obtained. For this purpose, we used quantitative real-time PCR (qPCR) and immunocytochemistry. The expression of mRNAs coding for all splicing isoforms of IGF-1 was observed in every tissue sample studied, with a significantly lower expression noted in the CRC as compared to the control samples. The cytoplasmic expression of IGF-1 protein was found in 50% of the CRC and in ~40% of the non-tumor tissues; however, no significant quantitative inter-group differences were observed. The expression of the IGF-1 gene in the 2 groups of tissues was controlled by the P1 and P2 promoters in a similar manner. No significant differences were detected in the expression of the IGF-1 A and B isoforms; however, their expression was significantly higher compared to that of isoform C. No significant differences were observed between the expression of Ki-67 mRNA in the CRC and control tissue even though the expression of the Ki-67 protein was higher in the CRC compared to the control samples. Ki-67 protein expression was associated with the macroscopic and microscopic aspects of CRC. A significant positive correlation was found between the local production of total mRNA and isoform A and the expression of Ki-67 mRNA, although only in the non-tumor tissues. In CRC samples, the local expression of the total IGF-1 mRNA and all splicing isoforms of IGF-1 m

  9. Most highly expressed protein-coding genes have a single dominant isoform.

    PubMed

    Ezkurdia, Iakes; Rodriguez, Jose Manuel; Carrillo-de Santa Pau, Enrique; Vázquez, Jesús; Valencia, Alfonso; Tress, Michael L

    2015-04-03

    Although eukaryotic cells express a wide range of alternatively spliced transcripts, it is not clear whether genes tend to express a range of transcripts simultaneously across cells, or produce dominant isoforms in a manner that is either tissue-specific or regardless of tissue. To date, large-scale investigations into the pattern of transcript expression across distinct tissues have produced contradictory results. Here, we attempt to determine whether genes express a dominant splice variant at the protein level. We interrogate peptides from eight large-scale human proteomics experiments and databases and find that there is a single dominant protein isoform, irrespective of tissue or cell type, for the vast majority of the protein-coding genes in these experiments, in partial agreement with the conclusions from the most recent large-scale RNAseq study. Remarkably, the dominant isoforms from the experimental proteomics analyses coincided overwhelmingly with the reference isoforms selected by two completely orthogonal sources, the consensus coding sequence variants, which are agreed upon by separate manual genome curation teams, and the principal isoforms from the APPRIS database, predicted automatically from the conservation of protein sequence, structure, and function.

  10. Postnatal expression of myosin isoforms in the genioglossus and diaphragm muscles.

    PubMed

    Brozanski, B S; Daood, M J; Watchko, J F; LaFramboise, W A; Guthrie, R D

    1993-04-01

    We studied the expression of myosin heavy chain (MHC) and native myosin isoforms in the genioglossus (GG) and costal diaphragm (DIA) muscles of the rat during postnatal development using both denaturing and nondenaturing gel electrophoresis. Primary myotubes in both fast and slow muscles homogeneously express slow as well as embryonic myosin. Since the adult GG is comprised primarily of fast MHC isoforms, whereas the adult DIA is characterized by a mixture of MHC slow and fast isoforms, we hypothesized that the GG and DIA would be subject to different temporal patterns of MHC isoform expression during postnatal development. Native myosin and MHC gels demonstrated a persistence of neonatal MHC (MHC neo) on day 25 in the GG, whereas this isoform was not detected beyond day 21 in the DIA. The MHC phenotype in GG of the adult demonstrated a predominance of MHC 2X (35% +/- 8) and MHC 2B (45% +/- 10) with a smaller proportion of MHC 2A (19% +/- 5). In contrast, the MHC phenotype in adult DIA was characterized by approximately equal proportions of MHC slow (25% +/- 3), MHC 2A (34% +/- 10), and MHC 2X (31% +/- 12) with a small percentage of MHC 2B (9% +/- 7). These data suggest that postnatal regulation of MHC expression in the GG and DIA is muscle specific.

  11. Differential gene expression of CYP3A isoforms in equine liver and intestines.

    PubMed

    Tydén, E; Löfgren, M; Pegolo, S; Capolongo, F; Tjälve, H; Larsson, P

    2012-12-01

    Recently, seven CYP3A isoforms - CYP3A89, CYP3A93, CYP3A94, CYP3A95, CYP3A96, CYP3A97 and CYP129 - have been isolated from the horse genome. In this study, we have examined the hepatic and intestinal gene expression of these CYP3A isoforms using TaqMan probes. We have also studied the enzyme activity using luciferin-isopropyl acetal (LIPA) as a substrate. The results show a differential gene expression of the CYP3A isoforms in the liver and intestines in horses. In the liver, CYP3A89, CYP3A94, CYP3A96 and CYP3A97 were highly expressed, while in the intestine there were only two dominating isoforms, CYP3A93 and CYP3A96. The isoform CYP3A129 was not detected in the liver or the intestine, although this gene consists of a complete set of exons and should therefore code for a functional protein. It is possible that this gene is expressed in tissues other than the liver and intestines. In the intestine, both CYP3A96 and CYP3A93 showed the highest gene expression in the duodenum and the proximal parts of the jejunum. This correlated with a high protein expression in these tissues. Studies of the enzyme activity showed the same K(m) for the LIPA substrate in the liver and the intestine, while the maximum velocity (V(max)) in the liver was higher than in the intestine. Our finding of a differential gene expression of the CYP3A isoforms in the liver and the intestines contributes to a better understanding of drug metabolism in horses.

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

    PubMed

    Ihnatovych, Ivanna; Sielski, Neil L; Hofmann, Wilma A

    2014-01-01

    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.

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

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    2004-01-01

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

  16. Drosophila tissues with different metamorphic responses to ecdysone express different ecdysone receptor isoforms.

    PubMed

    Talbot, W S; Swyryd, E A; Hogness, D S

    1993-07-02

    In D. melanogaster a pulse of the steroid hormone ecdysone triggers the larval-to-adult metamorphosis, a complex process in which this hormone induces imaginal tissues to generate adult structures and larval tissues to degenerate. We show that the EcR gene encodes three ecdysone receptor isoforms (EcR-A, EcR-B1, and EcR-B2) that have common DNA- and hormone-binding domains but different N-terminal regions. We have used isoform-specific monoclonal antibodies to show that at the onset of metamorphosis different ecdysone target tissues express different isoform combinations in a manner consistent with the proposition that the different metamorphic responses of these tissues require different combinations of the EcR isoforms. We have also determined temporal developmental profiles of the EcR isoforms and their mRNAs in whole animals, showing that different isoforms predominate at different developmental stages that are marked by a pulse of ecdysone.

  17. Pattern of FGF-2 isoform expression correlated with its biological action in experimental prolactinomas.

    PubMed

    Mukdsi, Jorge H; De Paul, Ana Louis; Petiti, Juan P; Gutiérrez, Silvina; Aoki, Agustín; Torres, Alicia I

    2006-10-01

    Fibroblast growth factor-2 (FGF-2) synthesized in the pituitary is involved in the formation and progression of pituitary tumors. The aim of this study was to analyze the pattern expression of two FGF-2 isoforms at different subcellular levels and to determine its correlation with prolactinoma development. Estrogen administration to male rats for 7, 20, and 60 days generated pituitary tumors, with lactotrophs being the prevalent cell type. Ultrastructural immunolabeling showed FGF-2 in the cytosolic and nuclear compartments of somatotrophs, lactotrophs and gonadotrophs, as well as in folliculo-stellate cells of normal rats. Estrogen stimulation increased FGF-2 immunoreactivity in various tumors and enhanced the expression of two FGF-2 isoforms, 18 and 22 kDa, as quantified by western blot. The 18 kDa isoform observed in cytosol extracts reached the highest levels after 60 days of hormonal stimulation and this was related to lactotroph proliferation. However, the 22 kDa FGF-2 isoform was only detected in the nuclear compartment and achieved the maximum expression at 7 days of estrogen treatment, without any correlation with lactotroph proliferation. These results suggest that the 18 kDa FGF-2 may play a role in the modulation of lactotroph proliferation in prolactinomas induced by estrogen. The overproduction of both FGF-2 isoforms appears to be implicated in autocrine-paracrine-intracrine mitogenic loops; this FGF-2 activity could lead to uncontrolled cell growth, angiogenesis, and tumor formation.

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

    SciTech Connect

    Lam, Le Thanh; Boehm, Sabrina V.; Roberts, Roland G.; Morris, Glenn E.

    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.

  19. Probe-Level Analysis of Expression Microarrays Characterizes Isoform-Specific Degradation during Mouse Oocyte Maturation

    PubMed Central

    Salisbury, Jesse; Hutchison, Keith W.; Wigglesworth, Karen; Eppig, John J.; Graber, Joel H.

    2009-01-01

    Background Gene expression microarrays have provided many insights into changes in gene expression patterns between different tissue types, developmental stages, and disease states. Analyses of these data focused primarily measuring the relative abundance of transcripts of a gene, while treating most or all transcript isoforms as equivalent. Differences in the selection between transcript isoforms can, however, represent critical changes to either the protein product or the posttranscriptional regulation of the transcript. Novel analyses on existing microarray data provide fresh insights and new interpretations into transcriptome-wide changes in expression. Methodology A probe-level analysis of existing gene expression arrays revealed differences in mRNA processing, primarily affecting the 3′-untranslated region. Working with the example of microarrays drawn from a transcriptionally silent period of mouse oocyte development, probe-level analysis (implemented here as rmodel) identified genes whose transcript isoforms have differing stabilities. Comparison of micorarrays measuring cDNA generated from oligo-dT and random primers revealed further differences in the polyadenylation status of some transcripts. Additional analysis provided evidence for sequence-targeted cleavage, including putative targeting sequences, as one mechanism of degradation for several hundred transcripts in the maturing oocyte. Conclusions The capability of probe-level analysis to elicit novel findings from existing expression microarray data was demonstrated. The characterization of differences in stability between transcript isoforms in maturing mouse oocytes provided some mechanistic details of degradation. Similar analysis of existing archives of expression microarray data will likely provide similar discoveries. PMID:19834616

  20. Bronchial Epithelial Cells from Asthmatic Patients Display Less Functional HLA-G Isoform Expression.

    PubMed

    Carlini, Federico; Picard, Christophe; Garulli, Céline; Piquemal, David; Roubertoux, Pierre; Chiaroni, Jacques; Chanez, Pascal; Gras, Delphine; Di Cristofaro, Julie

    2017-01-01

    Not all asthmatic patients adequately respond to current available treatments, such as inhaled corticosteroids or omalizumab(®). New treatments will aim to target the bronchial epithelium-immune response interaction using different pathways. HLA-G is involved in immunomodulation and may promote epithelial cell differentiation and proliferation. HLA-G protein has several isoforms generated by alternative splicing that might have differential functionalities. HLA-G protein expression and genetic polymorphisms have been reported to be associated with asthma. Our hypothesis is that bronchial epithelium from asthmatic patients displays less functional HLA-G isoforms. HLA-G transcriptional isoforms were quantified by real-time PCR in human bronchial epithelium cells (HBEC) grown in air-liquid interface culture obtained from five healthy controls (HC), seven patients with mild asthma (MA), and seven patients with severe asthma (SA). They were re-differentiated, and IL-13 exposure was used as a proxy for a pro-inflammatory cytokine. HLA-G protein expression was assessed by western blot analysis. HLA-G allele was typed by direct sequencing. Our results showed that both MA and SA display less functional HLA-G isoforms than HC (p < 0.05); in vitro HBEC re-differentiation from SA displays a particular isoform expression profile compared to MA and HC (p = 0.03); HLA-G*01:06 frequency in MA and SA was significantly higher than in the healthy population (p = 0.03 and p < 0.001, respectively); and IL-13 exposure had no impact on HLA-G expression. Our results support that an impaired expression of HLA-G isoforms in asthmatic patients could contribute to the loss of inflammation control and epithelium structural remodeling. Therefore, HLA-G might be an interesting alternative target for asthmatic patients not adequately responding to current drugs.

  1. Bronchial Epithelial Cells from Asthmatic Patients Display Less Functional HLA-G Isoform Expression

    PubMed Central

    Carlini, Federico; Picard, Christophe; Garulli, Céline; Piquemal, David; Roubertoux, Pierre; Chiaroni, Jacques; Chanez, Pascal; Gras, Delphine; Di Cristofaro, Julie

    2017-01-01

    Not all asthmatic patients adequately respond to current available treatments, such as inhaled corticosteroids or omalizumab®. New treatments will aim to target the bronchial epithelium–immune response interaction using different pathways. HLA-G is involved in immunomodulation and may promote epithelial cell differentiation and proliferation. HLA-G protein has several isoforms generated by alternative splicing that might have differential functionalities. HLA-G protein expression and genetic polymorphisms have been reported to be associated with asthma. Our hypothesis is that bronchial epithelium from asthmatic patients displays less functional HLA-G isoforms. HLA-G transcriptional isoforms were quantified by real-time PCR in human bronchial epithelium cells (HBEC) grown in air–liquid interface culture obtained from five healthy controls (HC), seven patients with mild asthma (MA), and seven patients with severe asthma (SA). They were re-differentiated, and IL-13 exposure was used as a proxy for a pro-inflammatory cytokine. HLA-G protein expression was assessed by western blot analysis. HLA-G allele was typed by direct sequencing. Our results showed that both MA and SA display less functional HLA-G isoforms than HC (p < 0.05); in vitro HBEC re-differentiation from SA displays a particular isoform expression profile compared to MA and HC (p = 0.03); HLA-G*01:06 frequency in MA and SA was significantly higher than in the healthy population (p = 0.03 and p < 0.001, respectively); and IL-13 exposure had no impact on HLA-G expression. Our results support that an impaired expression of HLA-G isoforms in asthmatic patients could contribute to the loss of inflammation control and epithelium structural remodeling. Therefore, HLA-G might be an interesting alternative target for asthmatic patients not adequately responding to current drugs. PMID:28303134

  2. PrPC, the cellular isoform of the human prion protein, is a novel biomarker of HIV-associated neurocognitive impairment and mediates neuroinflammation.

    PubMed

    Roberts, Toni K; Eugenin, Eliseo A; Morgello, Susan; Clements, Janice E; Zink, M Christine; Berman, Joan W

    2010-10-01

    Of the 33 million people infected with the human immunodeficiency virus (HIV) worldwide, 40-60% of individuals will eventually develop neurocognitive sequelae that can be attributed to the presence of HIV-1 in the central nervous system (CNS) and its associated neuroinflammation despite antiretroviral therapy. PrP(C) (protease resistant protein, cellular isoform) is the nonpathological cellular isoform of the human prion protein that participates in many physiological processes that are disrupted during HIV-1 infection. However, its role in HIV-1 CNS disease is unknown. We demonstrate that PrP(C) is significantly increased in both the CNS of HIV-1-infected individuals with neurocognitive impairment and in SIV-infected macaques with encephalitis. PrP(C) is released into the cerebrospinal fluid, and its levels correlate with CNS compromise, suggesting it is a biomarker of HIV-associated neurocognitive impairment. We show that the chemokine (c-c Motif) Ligand-2 (CCL2) increases PrP(C) release from CNS cells, while HIV-1 infection alters PrP(C) release from peripheral blood mononuclear cells. Soluble PrP(C) mediates neuroinflammation by inducing astrocyte production of both CCL2 and interleukin 6. This report presents the first evidence that PrP(C) dysregulation occurs in cognitively impaired HIV-1-infected individuals and that PrP(C) participates in the pathogenesis of HIV-1-associated CNS disease.

  3. Differential expression of two distinct functional isoforms of melanopsin (Opn4) in the mammalian retina.

    PubMed

    Pires, Susana S; Hughes, Steven; Turton, Michael; Melyan, Zare; Peirson, Stuart N; Zheng, Lei; Kosmaoglou, Maria; Bellingham, James; Cheetham, Michael E; Lucas, Robert J; Foster, Russell G; Hankins, Mark W; Halford, Stephanie

    2009-09-30

    Melanopsin is the photopigment that confers photosensitivity to a subset of retinal ganglion cells (pRGCs) that regulate many non-image-forming tasks such as the detection of light for circadian entrainment. Recent studies have begun to subdivide the pRGCs on the basis of morphology and function, but the origin of these differences is not yet fully understood. Here we report the identification of two isoforms of melanopsin from the mouse Opn4 locus, a previously described long isoform (Opn4L) and a novel short isoform (Opn4S) that more closely resembles the sequence and structure of rat and human melanopsins. Both isoforms, Opn4L and Opn4S, are expressed in the ganglion cell layer of the retina, traffic to the plasma membrane and form a functional photopigment in vitro. Quantitative PCR revealed that Opn4S is 40 times more abundant than Opn4L. The two variants encode predicted proteins of 521 and 466 aa and only differ in the length of their C-terminal tails. Antibodies raised to isoform-specific epitopes identified two discrete populations of melanopsin-expressing RGCs, those that coexpress Opn4L and Opn4S and those that express Opn4L only. Recent evidence suggests that pRGCs show a range of anatomical subtypes, which may reflect the functional diversity reported for mouse Opn4-mediated light responses. The distinct isoforms of Opn4 described in this study provide a potential molecular basis for generating this diversity, and it seems likely that their differential expression plays a role in generating the variety of pRGC light responses found in the mammalian retina.

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

    PubMed Central

    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

  5. Postnatal expression of myosin isoforms in an expiratory muscle--external abdominal oblique.

    PubMed

    Watchko, J F; Daood, M J; Vazquez, R L; Brozanski, B S; LaFramboise, W A; Guthrie, R D; Sieck, G C

    1992-11-01

    We studied the postnatal expression of heavy-chain (MHC) and native myosin isoforms in an expiratory abdominal muscle of the rat, the external abdominal oblique (EO). Moreover, we contrasted EO myosin expression with that of the costal diaphragm (DIA) to draw inspiratory vs. expiratory muscle comparisons during development. Examination of MHC gels demonstrated a mature phenotype of slow and adult fast myosin isoforms at an earlier age in the EO (day 60) than in the DIA [day > 115 (adult)]. The mature MHC phenotype of the EO was characterized by a preponderance of MHC 2B, whereas the DIA was characterized by approximately equal portions of MHC slow, MHC 2A, and MHC 2X. During early postnatal development, there was a delay in the expression of MHC 2A in the EO compared with the DIA. However, MHC 2B, expressed later in development in both muscles, was noted in the EO before the DIA. We conclude that 1) the EO mature myosin phenotype is characterized by a preponderance of fast myosin isoforms and 2) the EO and DIA muscles are subject to different temporal patterns of isoform expression during postnatal development.

  6. Dystrophin Dp71 Isoforms Are Differentially Expressed in the Mouse Brain and Retina: Report of New Alternative Splicing and a Novel Nomenclature for Dp71 Isoforms.

    PubMed

    Aragón, Jorge; González-Reyes, Mayram; Romo-Yáñez, José; Vacca, Ophélie; Aguilar-González, Guadalupe; Rendón, Alvaro; Vaillend, Cyrille; Montañez, Cecilia

    2017-01-27

    Multiple dystrophin Dp71 isoforms have been identified in rats, mice, and humans and in several cell line models. These Dp71 isoforms are produced by the alternative splicing of exons 71 to 74 and 78 and intron 77. Three main groups of Dp71 proteins are defined based on their C-terminal specificities: Dp71d, Dp71f, and Dp71e. Dp71 is highly expressed in the brain and retina; however, the specific isoforms present in these tissues have not been determined to date. In this work, we explored the expression of Dp71 isoforms in the mouse brain and retina using RT-PCR assays followed by the cloning of PCR products into the pGEM-T Easy vector, which was used to transform DH5α cells. Dp71-positive colonies were later analyzed by PCR multiplex and DNA sequencing to determine the alternative splicing. We thus demonstrated the expression of Dp71 transcripts corresponding to Dp71, Dp71a, Dp71c, Dp71b, Dp71ab, Dp71 Δ110, and novel Dp71 isoforms spliced in exon 74; 71 and 74; 71, 73 and 74; and 74 and 78, which we named Dp71d Δ74 , Dp71d Δ71,74 , Dp71d Δ71,73-74 , and Dp71f Δ74 , respectively. Additionally, we demonstrated that the Dp71d group of isoforms is highly expressed in the brain, while the Dp71f group predominates in the retina, at both the cDNA and protein levels. These findings suggest that distinct Dp71 isoforms may play different roles in the brain and retina.

  7. Differential expression and subcellular distribution of dystrophin Dp71 isoforms during differentiation process.

    PubMed

    Marquez, F G; Cisneros, B; Garcia, F; Ceja, V; Velázquez, F; Depardón, F; Cervantes, L; Rendón, A; Mornet, D; Rosas-vargas, H; Mustre, M; Montañez, C

    2003-01-01

    Dp71 is the major product of the Duchenne muscular dystrophy gene in the brain. In order to study the function of Dp71 in the nervous system we examined the expression of Dp71 isoforms in PC12 rat pheochromocytoma cell line, a well-established system to study neuronal differentiation. We show by reverse transcriptase-polymerase chain reaction and Western blot assays that PC12 cells express two Dp71 isoforms. One isoform lacks exon 71 and the other isoform lacks exons 71 and 78 (Dp71d and Dp71f isoforms respectively). Nerve growth factor-induced neuronal differentiation of PC12 cells results in differential regulation of the expression and subcellular localization of Dp71 isoforms: a) the amount of Dp71f protein increases nine-fold in total extracts while Dp71d increases up to seven-fold in nuclear extracts; b) Dp71f relocates from the cytoplasm to neuritic processes, being prominent at varicosities and the growth cone; c) Dp71d relocates almost entirely to the nucleus and is detected to a lower extent in the cytoplasm and neuritic processes. Dp71f co-localizes with beta-dystroglycan and synaptophysin while Dp71d co-localizes with beta-dystroglycan in the nucleus. Dp71d accumulates at cell-cell contacts where Dp71f is absent. These results suggest that Dp71d and Dp71f associate with different subcellular complexes and therefore may have distinct functions in PC12 cells.

  8. Human hepatocellular carcinoma expresses specific PCNA isoforms: an in vivo and in vitro evaluation.

    PubMed

    Venturi, Annamaria; Piaz, Fabrizio Dal; Giovannini, Catia; Gramantieri, Laura; Chieco, Pasquale; Bolondi, Luigi

    2008-09-01

    Proliferating cell nuclear antigen (PCNA) is a 36 kDa protein involved in several cellular mechanisms, including DNA synthesis and repair, cell cycle regulation and apoptosis. An alteration in PCNA structure might contribute to DNA-damage accumulation in cancer cells. This study was aimed to evaluate the PCNA pattern of expression, in terms of aggregation status, isoforms and post-translational modifications, in human hepatocellular carcinoma (HCC) and cirrhosis as well as in HCC cell lines. Twelve HCCs and surrounding cirrhotic tissues were analysed, along with HepG2, Hep3B and SNU-398 cell lines. Normal liver specimens and cirrhosis without HCC were included as controls. Both DNA-bound and DNA-unbound PCNA fractions were analysed, and PCNA pattern of expression was displayed on two-dimensional gel electrophoresis followed by western blot. Results were confirmed by mass spectrometry. To compare HCCs vs surrounding tissues, immunolabelling and immunostaining were performed. In 6 of 12 HCCs and in cell lines, we found three major PCNA acidic forms, corresponding to monomers, probably dimers and trimers, and a basic isoform. In the six remaining HCCs, only a PCNA acidic form associated with multiple basic isoforms was detected. Importantly, the PCNA basic form was not found in cirrhotic tissues. To clarify the nature of the detected PCNA isoforms, ubiquitin-specific immunoblotting as well as phosphatase treatment were employed. A PCNA-ubiquitylated form in cell lines and PCNA-phosphorylated isoforms in 6 of 12 HCCs were detected. Finally, in the DNA-bound fraction we detected only an acidic PCNA monomeric form. We conclude that human hepatocellular carcinoma expresses specific PCNA isoforms compared to those found in cirrhosis, implicating a role for PCNA functional alterations in hepatocarcinogenesis.

  9. Differential ICAM-1 Isoform Expression Regulates the Development and Progression of Experimental Autoimmune Encephalomyelitis

    PubMed Central

    Wohler, Jillian E.; Schoeb, Trenton R.; Bullard, Daniel C.

    2013-01-01

    Intercellular adhesion molecule-1 (ICAM-1) functions in leukocyte trafficking, activation, and the formation of the immunological synapse. ICAM-1 is a member of the immunoglobulin superfamily of adhesion proteins, which share a similar structure of repeating Ig-like domains. Many genes in this family, including ICAM-1, show alternative splicing leading to the production of different protein isoforms, although little functional information is available regarding the expression patterns, ligand interactions, and functions of these isoforms, especially those arising from the ICAM-1 gene. In this study, we show using different lines of mutant mice (Icam1tm1Jcgr and Icam1tm1Bay) that alterations in the expression of the alternatively spliced ICAM-1 isoforms can significantly influence the disease course during the development of EAE. Icam1tm1Jcgr mutant mice, unlike Icam1tm1Bay mutants, do not express isoforms containing the Mac-1 binding domain and had significantly attenuated of EAE. In contrast, Icam1tm1Bay mice developed severe EAE in both active and adoptive transfer models compared to both Icam1tm1Jcgr and wild type mice. We also observed that T cells from Icam1tm1Bay mice displayed increased proliferation kinetics and produced higher levels of IFN-γ compared to Icam1tm1Jcgr and wild type mice. Thus, our investigations show that the alternatively spliced ICAM-1 isoforms are functional, and play key roles during the progression of CNS inflammation and demyelination in EAE. Furthermore, our findings suggest that these isoforms may also play key roles in controlling the development of inflammatory diseases such as multiple sclerosis, possibly through differential engagement with ICAM-1 ligands such as Mac-1. PMID:20371120

  10. Differential ICAM-1 isoform expression regulates the development and progression of experimental autoimmune encephalomyelitis.

    PubMed

    Hu, Xianzhen; Barnum, Scott R; Wohler, Jillian E; Schoeb, Trenton R; Bullard, Daniel C

    2010-05-01

    Intercellular adhesion molecule-1 (ICAM-1) functions in leukocyte trafficking, activation, and the formation of the immunological synapse. ICAM-1 is a member of the immunoglobulin superfamily of adhesion proteins, which share a similar structure of repeating Ig-like domains. Many genes in this family, including ICAM-1, show alternative splicing leading to the production of different protein isoforms, although little functional information is available regarding the expression patterns, ligand interactions, and functions of these isoforms, especially those arising from the ICAM-1 gene. In this study, we show using different lines of mutant mice (Icam1(tm1Jcgr) and Icam1(tm1Bay)) that alterations in the expression of the alternatively spliced ICAM-1 isoforms can significantly influence the disease course during the development of EAE. Icam1(tm1Jcgr) mutant mice, unlike Icam1(tm1Bay) mutants, do not express isoforms containing the Mac-1 binding domain and had significantly attenuated of EAE. In contrast, Icam1(tm1Bay) mice developed severe EAE in both active and adoptive transfer models compared to both Icam1(tm1Jcgr) and wild type mice. We also observed that T cells from Icam1(tm1Bay) mice displayed increased proliferation kinetics and produced higher levels of IFN-gamma compared to Icam1(tm1Jcgr) and wild type mice. Thus, our investigations show that the alternatively spliced ICAM-1 isoforms are functional, and play key roles during the progression of CNS inflammation and demyelination in EAE. Furthermore, our findings suggest that these isoforms may also play key roles in controlling the development of inflammatory diseases such as multiple sclerosis, possibly through differential engagement with ICAM-1 ligands such as Mac-1.

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

    SciTech Connect

    Brizio, Carmen; Galluccio, Michele; Wait, Robin; Torchetti, Enza Maria; Bafunno, Valeria; Accardi, Rosita; Gianazza, Elisabetta; Indiveri, Cesare; Barile, Maria . E-mail: m.barile@biologia.uniba.it

    2006-06-09

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

  12. Differential expression of heat shock protein 90 isoforms in small cell lung cancer

    PubMed Central

    Lee, Ji Hyun; Kang, Kyung Woo; Kim, Jeong-Eun; Hwang, Sang Won; Park, Jae Hong; Kim, Seok-Hyun; Ji, Jun Ho; Kim, Tae Gyu; Nam, Hyun-Yeol; Roh, Mee Sook; Lee, Eun Hee; Park, Moon-il; Kim, Mee-Seon; Lee, Hyoun Wook

    2015-01-01

    Heat shock protein 90 (HSP90), a molecular chaperone, plays important roles in cellular protection against various stressful stimuli and in the regulation of cellular growth and apoptosis. HSP90 has 4 different types of human isoforms; HSP90α, HSP90β, glucose related protein 94 (GRP94) and tumor necrosis factor (TNF) receptor-associated protein 1 (TRAP1). We assessed the differential expression of these HSP90 isoforms in small-cell lung cancer (SCLC) and the correlation of their expression levels with clinicopathological factors and patient survival rates. This study included 117 SCLCs, comprised of 108 primary and 9 metastatic tumor tissues. We performed immunohistochemical staining for HSP90α, HSP90β, GRP94 and TRAP1 in 117 tumors and found that HSP90α and HSP90β were positive in 11 (9%) and 61 tumors (52%), respectively, most of which showed weak expression, whereas GRP94 and TRAP1 were positive in 115 (98%) and 117 tumors (100%), respectively, the majority of which showed moderate or strong expression. None of the HSP90 isoforms showed significant associations with clinicopathological factors or survival status in patients with SCLC. Our results indicate that GRP94 and TRAP1 might contribute more to the carcinogenesis or biology of SCLC than HSP90α and HSP90β, and that isoform selectivity should be considered when HSP90 inhibitors are studied or utilized for the treatment of SCLC. PMID:26464709

  13. The Impact of Endurance Training on Human Skeletal Muscle Memory, Global Isoform Expression and Novel Transcripts

    PubMed Central

    Lindholm, Maléne E; Giacomello, Stefania; Werne Solnestam, Beata; Kjellqvist, Sanela

    2016-01-01

    Regularly performed endurance training has many beneficial effects on health and skeletal muscle function, and can be used to prevent and treat common diseases e.g. cardiovascular disease, type II diabetes and obesity. The molecular adaptation mechanisms regulating these effects are incompletely understood. To date, global transcriptome changes in skeletal muscles have been studied at the gene level only. Therefore, global isoform expression changes following exercise training in humans are unknown. Also, the effects of repeated interventions on transcriptional memory or training response have not been studied before. In this study, 23 individuals trained one leg for three months. Nine months later, 12 of the same subjects trained both legs in a second training period. Skeletal muscle biopsies were obtained from both legs before and after both training periods. RNA sequencing analysis of all 119 skeletal muscle biopsies showed that training altered the expression of 3,404 gene isoforms, mainly associated with oxidative ATP production. Fifty-four genes had isoforms that changed in opposite directions. Training altered expression of 34 novel transcripts, all with protein-coding potential. After nine months of detraining, no training-induced transcriptome differences were detected between the previously trained and untrained legs. Although there were several differences in the physiological and transcriptional responses to repeated training, no coherent evidence of an endurance training induced transcriptional skeletal muscle memory was found. This human lifestyle intervention induced differential expression of thousands of isoforms and several transcripts from unannotated regions of the genome. It is likely that the observed isoform expression changes reflect adaptational mechanisms and processes that provide the functional and health benefits of regular physical activity. PMID:27657503

  14. Cloning and expression of the mouse histamine H3 receptor: evidence for multiple isoforms.

    PubMed

    Rouleau, Agnès; Héron, Anne; Cochois, Véronique; Pillot, Catherine; Schwartz, Jean-Charles; Arrang, Jean-Michel

    2004-09-01

    The existence of mouse H3-receptor isoforms was investigated by PCR analysis and cDNA cloning. Splicing mechanisms previously reported in various species are conserved in the mouse. The retention/deletion of a fragment in the third intracellular loop of the mouse receptor leads to the existence of three isoforms designated mH(3(445)), mH(3(413)) and mH(3(397)) according to the length of their deduced amino acid sequence. PCR analysis showed that mouse H3-receptor isoforms display different expression patterns in the brain. Following expression in Cos-1 cells, [125I]iodoproxyfan binding indicated similar pharmacological profiles of the mH(3(445)), mH(3(413)) and mH(3(397)) isoforms. The pharmacological profile of the mouse H3 receptor is more similar to the rat receptor than to the human receptor, although some differences were also observed between the mouse and rat receptors. For example, the potency of thioperamide and ciproxifan is slightly higher at the mouse receptor than at the rat receptor but 40-100-fold higher than at the human receptor. In situ hybridization histochemistry showed that the distribution of H3-receptor mRNAs in the mouse brain is rather similar to that previously reported in the rat brain. However, the autoradiographic and cellular expression patterns observed in several brain areas such as the thalamus or hippocampus reveal important differences between the two species.

  15. Transgenic Rabbits Expressing Ovine PrP Are Susceptible to Scrapie

    PubMed Central

    Sarradin, Pierre; Viglietta, Céline; Limouzin, Claude; Andréoletti, Olivier; Daniel-Carlier, Nathalie; Barc, Céline; Leroux-Coyau, Mathieu; Berthon, Patricia; Chapuis, Jérôme; Rossignol, Christelle; Gatti, Jean-Luc; Belghazi, Maya; Labas, Valérie; Vilotte, Jean-Luc; Béringue, Vincent; Lantier, Frédéric; Laude, Hubert; Houdebine, Louis-Marie

    2015-01-01

    Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases affecting a wide range of mammalian species. They are caused by prions, a proteinaceous pathogen essentially composed of PrPSc, an abnormal isoform of the host encoded cellular prion protein PrPC. Constrained steric interactions between PrPSc and PrPC are thought to provide prions with species specificity, and to control cross-species transmission into other host populations, including humans. Transgenetic expression of foreign PrP genes has been successfully and widely used to overcome the recognized resistance of mouse to foreign TSE sources. Rabbit is one of the species that exhibit a pronounced resistance to TSEs. Most attempts to infect experimentally rabbit have failed, except after inoculation with cell-free generated rabbit prions. To gain insights on the molecular determinants of the relative resistance of rabbits to prions, we generated transgenic rabbits expressing the susceptible V136R154Q171 allele of the ovine PRNP gene on a rabbit wild type PRNP New Zealand background and assessed their experimental susceptibility to scrapie prions. All transgenic animals developed a typical TSE 6–8 months after intracerebral inoculation, whereas wild type rabbits remained healthy more than 700 days after inoculation. Despite the endogenous presence of rabbit PrPC, only ovine PrPSc was detectable in the brains of diseased animals. Collectively these data indicate that the low susceptibility of rabbits to prion infection is not enciphered within their non-PrP genetic background. PMID:26248157

  16. Identification of a novel CoA synthase isoform, which is primarily expressed in Brain

    SciTech Connect

    Nemazanyy, Ivan . E-mail: nemazanyy@imbg.org.ua; Panasyuk, Ganna; Breus, Oksana; Zhyvoloup, Alexander; Filonenko, Valeriy; Gout, Ivan T. . E-mail: i.gout@ucl.ac.uk

    2006-03-24

    CoA and its derivatives Acetyl-CoA and Acyl-CoA are important players in cellular metabolism and signal transduction. CoA synthase is a bifunctional enzyme which mediates the final stages of CoA biosynthesis. In previous studies, we have reported molecular cloning, biochemical characterization, and subcellular localization of CoA synthase (CoASy). Here, we describe the existence of a novel CoA synthase isoform, which is the product of alternative splicing and possesses a 29aa extension at the N-terminus. We termed it CoASy {beta} and originally identified CoA synthase, CoASy {alpha}. The transcript specific for CoASy {beta} was identified by electronic screening and by RT-PCR analysis of various rat tissues. The existence of this novel isoform was further confirmed by immunoblot analysis with antibodies directed to the N-terminal peptide of CoASy {beta}. In contrast to CoASy {alpha}, which shows ubiquitous expression, CoASy {beta} is primarily expressed in Brain. Using confocal microscopy, we demonstrated that both isoforms are localized on mitochondria. The N-terminal extension does not affect the activity of CoA synthase, but possesses a proline-rich sequence which can bring the enzyme into complexes with signalling proteins containing SH3 or WW domains. The role of this novel isoform in CoA biosynthesis, especially in Brain, requires further elucidation.

  17. Expression of tropomyosin 2 gene isoforms in human breast cancer cell lines

    PubMed Central

    DUBE, SYAMALIMA; THOMAS, ANISH; ABBOTT, LYNN; BENZ, PATRICIA; MITSCHOW, CHARLES; DUBE, DIPAK K.; POIESZ, BERNARD J.

    2016-01-01

    In humans, four tropomyosin genes (TPM1, TPM2, TPM3, and TPM4) are known to produce a multitude of isoforms via alternate splicing and/or using alternate promoters. Expression of tropomyosin has been shown to be modulated at both the transcription and the translational levels. Tropomyosins are known to make up some of the stress fibers of human epithelial cells and differences in their expression has been demonstrated in malignant breast epithelial cell lines compared to 'normal' breast cell lines. We have recently reported the expression of four novel TPM1 isoforms (TPM1λ, TPM1µ, TPM1ν, and TPM1ξ) from human malignant tumor breast cell lines that are not expressed in adult and fetal cardiac tissue. Also, we evaluated their expression in relation to the stress fiber formation. In this study, nine malignant breast epithelial cell lines and three 'normal' breast cell lines were examined for stress fiber formation and expression of tropomyosin 2 (TPM2) isoform-specific RNAs and proteins. Stress fiber formation was assessed by immunofluorescence using Leica AF6000 Deconvolution microscope. Stress fiber formation was strong (++++) in the 'normal' cell lines and varied among the malignant cell lines (negative to +++). No new TPM2 gene RNA isoforms were identified, and TPM2β was the most frequently expressed TPM2 RNA and protein isoform. Stress fiber formation positively correlated with TPM2β RNA or protein expression at high, statistically significant degrees. Previously, we had shown that TPM1δ and TPM1λ positively and inversely, respectively, correlated with stress fiber formation. The most powerful predictor of stress fiber formation was the combination of TPM2β RNA, TPM1δ RNA, and the inverse of TPM1λ RNA expression. Our results suggest that the increased expression of TPM1λ and the decreased expression of TPM1δ RNA and TPM2β may lead to decreased stress fiber formation and malignant transformation in human breast epithelial cells. PMID:27108600

  18. Isoform-specific degradation of PR-B by E6-AP is critical for normal mammary gland development.

    PubMed

    Ramamoorthy, Sivapriya; Dhananjayan, Sarath C; Demayo, Francesco J; Nawaz, Zafar

    2010-11-01

    E6-associated protein (E6-AP), which was originally identified as an ubiquitin-protein ligase, also functions as a coactivator of estrogen (ER-α) and progesterone (PR) receptors. To investigate the in vivo role of E6-AP in mammary gland development, we generated transgenic mouse lines that either overexpress wild-type (WT) human E6-AP (E6-AP(WT)) or ubiquitin-protein ligase-defective E6-AP (E6-AP(C833S)) in the mammary gland. Here we show that overexpression of E6-AP(WT) results in impaired mammary gland development. In contrast, overexpression of E6-AP(C833S) or loss of E6-AP (E6-AP(KO)) increases lateral branching and alveolus-like protuberances in the mammary gland. We also show that the mammary phenotypes observed in the E6-AP transgenic and knockout mice are due, in large part, to the alteration of PR-B protein levels. We also observed alteration in ER-α protein level, which might contribute to the observed mammary phenotype by regulating PR expression. Furthermore, E6-AP regulates PR-B protein levels via the ubiquitin-proteasome pathway. Additionally, we also show that E6-AP impairs progesterone-induced Wnt-4 expression by decreasing the steady state level of PR-B in both mice and in human breast cancer cells. In conclusion, we present the novel observation that E6-AP controls mammary gland development by regulating PR-B protein turnover via the ubiquitin proteasome pathway. For the first time, we show that the E3-ligase activity rather than the coactivation function of E6-AP plays an important role in the mammary gland development, and the ubiquitin-dependent PR-B degradation is not required for its transactivation functions. This mechanism appears to regulate normal mammogenesis, and dysregulation of this process may be an important contributor to mammary cancer development and progression.

  19. Differential expression of endoglin in human melanoma cells expressing the V3 isoform of versican by microarray analysis.

    PubMed

    Miquel-Serra, Laia; Hernandez, Daniel; Docampo, María Jose; Bassols, Anna

    2010-01-01

    Versican is a large chondroitin sulfate proteoglycan produced by several tumor types, including malignant melanoma, which exists as four different splice variants. The large isoforms V0 and V1 promote melanoma cell proliferation. We previously described that overexpression of the short V3 isoform in MeWo human melanoma cells markedly reduced tumor cell growth in vitro and in vivo, but favored the appearance of secondary tumors. This study aimed to elucidate the mechanisms of V3 by identifying differentially expressed genes between parental and V3-expressing MeWo melanoma cells using microarray analysis. V3 expression significantly reduced the expression of endoglin, a transforming growth factor-β superfamily co-receptor. Other differentially expressed genes were VEGF and PPP1R14B. Changes in endoglin levels were validated by qRT-PCR and Western blotting.

  20. Plectin isoform 1-dependent nuclear docking of desmin networks affects myonuclear architecture and expression of mechanotransducers

    PubMed Central

    Staszewska, Ilona; Fischer, Irmgard; Wiche, Gerhard

    2015-01-01

    Plectin is a highly versatile cytoskeletal protein that acts as a mechanical linker between intermediate filament (IF) networks and various cellular structures. The protein is crucial for myofiber integrity. Its deficiency leads to severe pathological changes in skeletal muscle fibers of patients suffering from epidermolysis bullosa simplex with muscular dystrophy (EBS-MD). Skeletal muscle fibers express four major isoforms of plectin which are distinguished solely by alternative, relatively short, first exon-encoded N-terminal sequences. Each one of these isoforms is localized to a different subcellular compartment and plays a specific role in maintaining integrity and proper function(s) of myofibers. The unique role of individual isoforms is supported by distinct phenotypes of isoform-specific knockout mice and recently discovered mutations in first coding exons of plectin that lead to distinct, tissue-specific, pathological abnormalities in humans. In this study, we demonstrate that the lack of plectin isoform 1 (P1) in myofibers of mice leads to alterations of nuclear morphology, similar to those observed in various forms of MD. We show that P1-mediated targeting of desmin IFs to myonuclei is essential for maintenance of their typically spheroidal architecture as well as their proper positioning and movement along the myofiber. Furthermore, we show that P1 deficiency affects chromatin modifications and the expression of genes involved in various cellular functions, including signaling pathways mediating mechanotransduction. Mechanistically, P1 is shown to specifically interact with the myonuclear membrane-associated (BAR domain-containing) protein endophilin B. Our results open a new perspective on cytoskeleton-nuclear crosstalk via specific cytolinker proteins. PMID:26487297

  1. Expression and membrane localization of MCT isoforms along the length of the human intestine.

    PubMed

    Gill, Ravinder K; Saksena, Seema; Alrefai, Waddah A; Sarwar, Zaheer; Goldstein, Jay L; Carroll, Robert E; Ramaswamy, Krishnamurthy; Dudeja, Pradeep K

    2005-10-01

    Recent studies from our laboratory and others have demonstrated the involvement of monocarboxylate transporter (MCT)1 in the luminal uptake of short-chain fatty acids (SCFAs) in the human intestine. Functional studies from our laboratory previously demonstrated kinetically distinct SCFA transporters on the apical and basolateral membranes of human colonocytes. Although apical SCFA uptake is mediated by the MCT1 isoform, the molecular identity of the basolateral membrane SCFA transporter(s) and whether this transporter is encoded by another MCT isoform is not known. The present studies were designed to assess the expression and membrane localization of different MCT isoforms in human small intestine and colon. Immunoblotting was performed with the purified apical and basolateral membranes from human intestinal mucosa obtained from organ donor intestine. Immunohistochemistry studies were done on paraffin-embedded sections of human colonic biopsy samples. Immunoblotting studies detected a protein band of approximately 39 kDa for MCT1, predominantly in the apical membranes. The relative abundance of MCT1 mRNA and protein increased along the length of the human intestine. MCT4 (54 kDa) and MCT5 (54 kDa) isoforms showed basolateral localization and were highly expressed in the distal colon. Immunohistochemical studies confirmed that human MCT1 antibody labeling was confined to the apical membranes, whereas MCT5 antibody staining was restricted to the basolateral membranes of the colonocytes. We speculate that distinct MCT isoforms may be involved in SCFA transport across the apical or basolateral membranes in polarized colonic epithelial cells.

  2. Functional protein expression of multiple sodium channel alpha- and beta-subunit isoforms in neonatal cardiomyocytes.

    PubMed

    Kaufmann, Susann G; Westenbroek, Ruth E; Zechner, Christoph; Maass, Alexander H; Bischoff, Sebastian; Muck, Jenny; Wischmeyer, Erhard; Scheuer, Todd; Maier, Sebastian K G

    2010-01-01

    Voltage-gated sodium channels are composed of pore-forming alpha- and auxiliary beta-subunits and are responsible for the rapid depolarization of cardiac action potentials. Recent evidence indicates that neuronal tetrodotoxin (TTX) sensitive sodium channel alpha-subunits are expressed in the heart in addition to the predominant cardiac TTX-resistant Na(v)1.5 sodium channel alpha-subunit. These TTX-sensitive isoforms are preferentially localized in the transverse tubules of rodents. Since neonatal cardiomyocytes have yet to develop transverse tubules, we determined the complement of sodium channel subunits expressed in these cells. Neonatal rat ventricular cardiomyocytes were stained with antibodies specific for individual isoforms of sodium channel alpha- and beta-subunits. alpha-actinin, a component of the z-line, was used as an intracellular marker of sarcomere boundaries. TTX-sensitive sodium channel alpha-subunit isoforms Na(v)1.1, Na(v)1.2, Na(v)1.3, Na(v)1.4 and Na(v)1.6 were detected in neonatal rat heart but at levels reduced compared to the predominant cardiac alpha-subunit isoform, Na(v)1.5. Each of the beta-subunit isoforms (beta1-beta4) was also expressed in neonatal cardiac cells. In contrast to adult cardiomyocytes, the alpha-subunits are distributed in punctate clusters across the membrane surface of neonatal cardiomyocytes; no isoform-specific subcellular localization is observed. Voltage clamp recordings in the absence and presence of 20 nM TTX provided functional evidence for the presence of TTX-sensitive sodium current in neonatal ventricular myocardium which represents between 20 and 30% of the current, depending on membrane potential and experimental conditions. Thus, as in the adult heart, a range of sodium channel alpha-subunits are expressed in neonatal myocytes in addition to the predominant TTX-resistant Na(v)1.5 alpha-subunit and they contribute to the total sodium current.

  3. Gene Expression of Dnmt1 Isoforms in Porcine Oocytes, Embryos, and Somatic Cells

    PubMed Central

    DeCourcy, Kristi; Ball, Suyapa F.; Hylan, Darin; Ayares, David L.

    2013-01-01

    Abstract In the mouse, the dynamics of genomic methylation and the initial events of gametic imprinting are controlled by the activity of an oocyte isoform of the DNA methyltransferase-1 (Dnmt1o) enzyme. The objectives of this study were to identify the alternative splicing variants of Dnmt1 in porcine oocytes and determine the gene expression pattern of the different Dnmt1 isoforms during embryo development. A rapid amplification of cDNA ends (RACE ) system was used to amplify the 5′ cDNA end of Dnmt1 isoforms in porcine oocytes. RNA levels of the Dnmt1 isoforms were analyzed in porcine oocytes and embryos. DNMT1 protein expression of oocytes and somatic cells were analyzed by western blot and immunostaining. Two new Dnmt1o RNA isoforms were identified—Dnmt1o1 and Dnmt1o2. The previously reported somatic Dnmt1 isoform (Dnmt1s) was expressed at low but constant levels in oocytes and embryos from the two-cell to the blastocyst stage. Abundant RNA levels of Dnmt1o1 and Dnmt1o2 were detected in oocytes and embryos from the two- to the eight- to 16-cell stage. Levels of these Dnmt1o transcripts were low at the morula and blastocyst stages. Although Dnmt1s was present in all the somatic cell types analyzed, Dnmt1o1 and Dnmt1o2 were not detected in any somatic tissues. As predicted by the RNA sequence and verified by western blot analysis, Dnmt1o1 and Dnmt1o2 RNAs translate one DNMT1o enzyme. Western blot analysis confirmed that both the oocyte and the somatic forms of DNMT1 protein are present in porcine oocytes and early embryos, whereas somatic cells produce only DNMT1s protein. DNMT1o is localized mainly in the nuclei of oocytes and early embryos, whereas DNMT1s is expressed in the ooplasm cortex of oocytes and cytoplasm of early embryos. PMID:23808878

  4. Isoform composition, gene expression and sarcomeric protein phosphorylation in striated muscle of mice after space flight

    NASA Astrophysics Data System (ADS)

    Vikhlyantsev, Ivan; Ulanova, Anna; Salmov, Nikolay; Gritsyna, Yulia; Bobylev, Alexandr; Rogachevsky, Vadim; Shenkman, Boris; Podlubnaya, Zoya

    Using RT-PCR and SDS-PAGE, changes in isoform composition, gene expression, titin and nebulin phosphorylation, as well as changes in isoform composition of myosin heavy chains in striated muscles of mice were studied after 30-day-long space flight onboard the Russian spacecraft “BION-M” No. 1. The muscle fibre-type shift from slow-to-fast was observed in m. gastrocnemius and m. tibialis anterior of animals from “Flight” group. A decrease in the content of the NT and N2A titin isoforms and nebulin in the skeletal muscles of animals from “Flight” group was found. Meanwhile, significant differences in gene expression of these proteins in skeletal muscles of mice from “Flight” and “Control” groups were not observed. Using Pro-Q Diamond stain, an increase in titin phosphorylation in m. gastrocnemius of mice from “Flight” group was detected. The content of the NT, N2BA and N2B titin isoforms in cardiac muscle of mice from “Flight” and “Control” groups did not differ, nevertheless an increase in titin gene expression in the myocardium of the “Flight” group animals was found. The observed changes will be discussed in the context of theirs role in contractile activity of striated muscles of mice in conditions of weightlessness. This work was supported by the Russian Foundation for Basic Research (grants No. 14-04-32240, 14-04-00112). Acknowledgement. We express our gratitude to the teams of Institute of Biomedical Problems RAS and “PROGRESS” Corporation involved in the preparation of the “BION-M” mission.

  5. Expression of TGF-betas in the embryonic nervous system: analysis of interbalance between isoforms.

    PubMed

    Mecha, M; Rabadán, M A; Peña-Melián, A; Valencia, M; Mondéjar, T; Blanco, M J

    2008-06-01

    Transforming growth factor-beta (TGF-beta) is a family of growth factors with essential and multiple roles during embryonic development. In mammals, three isoforms (TGF-beta1, TGF-beta2, TGF-beta3) have been described. In the nervous system, the presence of TGF-beta1 has remained undetectable in other structures than meninges and choroids plexus, while TGF-beta2 and TGF-beta3 were considered as the neural members of the family. In the present study, we have analysed the expression pattern of the three isoforms in the neural tube, brain, and spinal cord during development in both mouse and chicken. The data reveal specific patterns for each isoform. This work also shows that both TGF-beta1 and TGF-beta3 are expressed in neural crest cells. In addition, we demonstrate the existence of interbalance between TGF-beta1 and TGF-beta3 with possible functional implications, which, together with the expression of TGF-beta1 in the CNS, represents one of the most important contributions of this work.

  6. The slow isoform of Xenopus troponin I is expressed in developing skeletal muscle but not in the heart.

    PubMed

    Warkman, Andrew S; Atkinson, Burr G

    2002-07-01

    In birds and mammals three isoforms of troponin I (TnI) exist; a slow (TnIs), a fast (TnIf) and a cardiac (TnIc). Although each of these isoforms is expressed in the adult forms of these organisms in a muscle fiber-type-specific manner, the gene encoding TnIs is also expressed within the developing heart of these vertebrates. Herein, our results demonstrate that the developing heart of Xenopus laevis, unlike its counterpart in birds and mammals, does not express the gene encoding the TnIs isoform and that the expression of this gene, as well as the one encoding the Xenopus TnIf isoform, is restricted to skeletal muscle.

  7. Mice exclusively expressing the short isoform of Smad2 develop normally and are viable and fertile

    PubMed Central

    Dunn, N. Ray; Koonce, Chad H.; Anderson, Dorian C.; Islam, Ayesha; Bikoff, Elizabeth K.; Robertson, Elizabeth J.

    2005-01-01

    Smad2 and Smad3 are closely related effectors of TGFβ/Nodal/Activin-related signaling. Smad3 mutant mice develop normally, whereas Smad2 plays an essential role in patterning the embryonic axis and specification of definitive endoderm. Alternative splicing of Smad2 exon 3 gives rise to two distinct protein isoforms. The short Smad2(Δexon3) isoform, unlike full-length Smad2, Smad2(FL), retains DNA-binding activity. Here, we show that Smad2(FL) and Smad2(Δexon3) are coexpressed throughout mouse development. Directed expression of either Smad2(Δexon3) or Smad3, but not Smad2(FL), restores the ability of Smad2-deficient embryonic stem (ES) cells to contribute descendants to the definitive endoderm in wild-type host embryos. Mice engineered to exclusively express Smad2(Δexon3) correctly specify the anterior–posterior axis and definitive endoderm, and are viable and fertile. Moreover, introducing a human Smad3 cDNA into the mouse Smad2 locus similarly rescues anterior–posterior patterning and definitive endoderm formation and results in adult viability. Collectively, these results demonstrate that the short Smad2(Δexon3) isoform or Smad3, but not full-length Smad2, activates all essential target genes downstream of TGFβ-related ligands, including those regulated by Nodal. PMID:15630024

  8. Energy balance-dependent regulation of ovine glucose 6-phosphate dehydrogenase protein isoform expression.

    PubMed

    Triantaphyllopoulos, Kostas A; Laliotis, George P; Bizelis, Iosif A

    2014-01-01

    G6PDH is the rate-limiting enzyme of the pentose phosphate pathway and one of the principal source of NADPH, a major cellular reductant. Importantly, in ruminant's metabolism the aforementioned NADPH provided, is utilized for de novo fatty acid synthesis. Previous work of cloning the ovine (Ovis aries) og6pdh gene has revealed the presence of two cDNA transcripts (og6pda and og6pdb), og6pdb being a product of alternative splicing not similar to any other previously reported.(1) In the current study the effect of energy balance in the ovine G6PDH protein expression was investigated, shedding light on the biochemical features and potential physiological role of the oG6PDB isoform. Changes in energy balance leads to protein expression changes in both transcripts, to the opposite direction and not in a proportional way. Negative energy balance was not in favor of the presence of any particular isoform, while both protein expression levels were not significantly different (P > 0.05). In contrast, at the transition point from negative to positive and on the positive energy balance, there is a significant increase of oG6PDA compared with oG6PDB protein expression (P < 0.001). Both oG6PDH protein isoforms changed significantly toward the positive energy balance. oG6PDA is escalating, while oG6PDB is falling, under the same stimulus (positive energy balance alteration). This change is also positively associated with increasing levels in enzyme activity, 4 weeks post-weaning in ewes' adipose tissue. Furthermore, regression analysis clearly demonstrated the linear correlation of both proteins in response to the WPW, while energy balance, enzyme activity, and oG6PDA relative protein expression follow the same escalating trend; in contrast, oG6PDB relative protein expression falls in time, similar to both transcripts accumulation pattern, as reported previously.(2.)

  9. New brain-specific beta-synuclein isoforms show expression ratio changes in Lewy body diseases.

    PubMed

    Beyer, Katrin; Munoz-Marmol, Ana M; Sanz, Carolina; Marginet-Flinch, Ruth; Ferrer, Isidro; Ariza, Aurelio

    2012-02-01

    Lewy body diseases (LBDs) include dementia with Lewy bodies (DLB) and Parkinson disease (PD). Alpha-synuclein (AS) aggregation is a key event in the pathogenesis of LBDs and beta-synuclein (BS) inhibits AS aggregation in vitro and in vivo. Recently, BS has been shown to interact directly with AS regulating its functionality and preventing its oligomerization, and a molecular subgroup of pure DLB lacks BS in cortical regions. In this study, we characterized four new BS transcript variants and analyzed their expression in neuronal and non-neuronal tissue, and their differential expression in frozen samples of three areas from brains of patients with pure Lewy body pathology (LBP), common LBP, Alzheimer pathology, and of controls. Relative mRNA expression was determined by real-time PCR with neuron-specific enolase 2 and synaptophysin as housekeeping genes, and expression changes were evaluated by the ΔΔCt method. Two main findings are in concordance with earlier studies. First, all BS isoforms are drastically diminished in the cortex of patients with pure LBP that had presented clinically as DLB but not PD with dementia. Second, an important shift of the isoform expression ratio was observed in the temporal cortex of all LBD cases, and the minor isoforms, normally absent in the midbrain, were detected in the caudate nucleus of all DLB samples. Our results provide further evidence for the role of minor transcript variants in the development of complex diseases and provide new insights into the pathogenesis of LBDs that may be important for the understanding of molecular mechanisms involved in these complex diseases.

  10. Influence of temperature, hypercapnia, and development on the relative expression of different hemocyanin isoforms in the common cuttlefish Sepia officinalis.

    PubMed

    Strobel, Anneli; Hu, Marian Y A; Gutowska, Magdalena A; Lieb, Bernhard; Lucassen, Magnus; Melzner, Frank; Pörtner, Hans O; Mark, Felix C

    2012-12-01

    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 acclimation to elevated seawater pCO(2) and temperature. Using quantitative PCR, we measured relative mRNA expression levels of three different hemocyanin isoforms in several ontogenetic stages (embryos, hatchlings, juveniles, and adults), under different temperatures and elevated seawater pCO(2). Our results indicate moderately altered hemocyanin expression in all embryonic stages acclimated to higher pCO(2), while hemocyanin expression in hatchlings and juveniles remained unaffected. During the course of development, total hemocyanin expression increased independently of pCO(2) or thermal acclimation status. Expression of isoform 3 is reported for the first time in a cephalopod in this study and was found to be generally low but highest in the embryonic stages (0.2% of total expression). Despite variable hemocyanin expression, hemolymph total protein concentrations remained constant in the experimental groups. Our data provide first evidence that ontogeny has a stronger influence on hemocyanin isoform expression than the environmental conditions chosen, and they suggest that hemocyanin protein abundance in response to thermal acclimation is regulated by post-transcriptional/translational rather than by transcriptional modifications.

  11. Expression of VEGF(xxx)b, the inhibitory isoforms of VEGF, in malignant melanoma.

    PubMed

    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) P<0.001 metastatic vs nonmetastatic), irrespective of tumour thickness, while the surrounding epidermis showed no difference in expression. Staining for total VEGF expression showed staining in metastatic and nonmetastatic melanomas, and normal epidermis. An absence of VEGF(xxx)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.

  12. Differences in Spatial Expression between 14-3-3 Isoforms in Germinating Barley Embryos1

    PubMed Central

    Testerink, Christa; van der Meulen, René M.; Oppedijk, Berry J.; de Boer, Albertus H.; Heimovaara-Dijkstra, Sjoukje; Kijne, Jan W.; Wang, Mei

    1999-01-01

    The family of 14-3-3 proteins is ubiquitous in eukaryotes and has been shown to exert an array of functions. We were interested in the possible role of 14-3-3 proteins in seed germination. Therefore, we studied the expression of 14-3-3 mRNA and protein in barley (Hordeum distichum L.) embryos during germination. With the use of specific cDNA probes and antibodies, we could detect individual expression of three 14-3-3 isoforms, 14-3-3A, 14-3-3B, and 14-3-3C. Each homolog was found to be expressed in barley embryos. Whereas protein levels of all three isoforms were constant during germination, mRNA expression was found to be induced upon imbibition of the grains. The induction of 14-3-3A gene expression during germination was different from that of 14-3-3B and 14-3-3C. In situ immunolocalization analysis showed similar spatial expression for 14-3-3A and 14-3-3B, while 14-3-3C expression was markedly different. Whereas 14-3-3A and 14-3-3B were expressed throughout the embryo, 14-3-3C expression was tissue specific, with the strongest expression observed in the scutellum and the L2 layer of the shoot apical meristem. These results show that 14-3-3 homologs are differently regulated in barley embryos, and provide a first step in acquiring more knowledge about the role of 14-3-3 proteins in the germination process. PMID:10482663

  13. DIFERENTIALLY EXPRESSED ADENYLYL CYCLASE ISOFORMS MEDIATE SECRETORY FUNCTIONS IN CHOLANGIOCYTE SUBPOPULATION

    PubMed Central

    Strazzabosco, Mario; Fiorotto, Romina; Melero, Saida; Glaser, Shannon; Francis, Heather; Spirlì, Carlo; Alpini, Gianfranco

    2009-01-01

    cAMP is generated by adenylyl cyclases (ACs) a group of enzymes with different tissue specificity and regulation. We hypothesized that AC isoforms are heterogeneously expressed along the biliary tree, are associated with specific secretory stimuli and are differentially modulated in cholestasis. Methods: Small (SDC) and large (LDC) cholangiocytes were isolated from controls and from lipopolysaccharide-treated (LPS) or α-naphthylisothiocyanate-treated (ANIT) rats. ACs isoforms expression was assessed by real-time PCR. Secretion and cAMP levels were measured in intrahepatic bile duct units after stimulation with secretin, forskolin, HCO3−/CO2, cholinergic and β-adrenergic agonists, with or without selected inhibitors or after silencing of AC8 or sAC with siRNA. Results: Gene expression of the Ca2+-insensitive isoforms (AC4, AC7) was higher in SDC, while that of the Ca2+-inhibitable (AC5, AC6, AC9), the Ca2+/calmodulin stimulated AC8, and the soluble sAC, was higher in LDC. Ca2+/calmodulin-inhibitors and AC8 gene silencing inhibited choleresis and cAMP production stimulated by secretin and acetylcholine, but not by forskolin. Secretion stimulated by isoproterenol and calcineurin-inibitors was cAMP-dependent and GABA-inhibitable, consistent with activation of AC9. Cholangiocyte secretion stimulated by isohydric changes in [HCO3−]i, was cAMP-dependent and inhibited by sAC-inhibitior and by sAC gene silencing. Treatment with LPS or ANIT increased expression of AC7 and sAC, while decreasing that of the others ACs. Conclusion: These studies demonstrate a previously unrecognized role of AC in biliary pathophysiology. In fact: 1) ACs isoforms are differentially expressed in cholangiocyte subpopulations, 2) AC8, AC9, and sAC mediate cholangiocyte secretion in response to secretin, β-adrenergic agonists, or changes in [HCO3−]i, respectively, 3) ACs gene expression is modulated in experimental cholestasis. PMID:19444869

  14. Structural Organization and Expression Pattern of the Canine RPGRIP1 Isoforms in Retinal Tissue

    PubMed Central

    Kuznetsova, Tatyana; Zangerl, Barbara; Goldstein, Orly; Acland, Gregory M.

    2011-01-01

    Purpose. To examine the structure and expression of RPGRIP1 in dog retina. Methods. Determination of the structural analysis and expression pattern of canine RPGRIP1 (cRPGRIP1) was based on cDNA amplification. Absolute quantification of the expression level of cRPGRIP1 splice variants was determined by qRT-PCR. Regulatory structures were examined by computational analysis of comparative genomics. Results. cRPGRIP1 encompasses 25 exons that harbor a 3627-bp open reading frame (ORF) encoding a 1209-amino-acid (aa)–predicted protein. In addition to the main transcript, five full-length and several partial cRPGRIP1 isoforms were identified revealing four alternative 3′-terminal exons—24, 19a, 19c, and 19d—three of which could potentially produce C-terminally truncated proteins that lack the RPGR-interacting domain. A complex organization of the 5′-UTR for the cRPGRIP1 splice products have been described, with a common promoter driving multiple isoforms, including four full-length transcripts using the 3′-terminal exon 24. In addition, a potential alternative internal promoter was revealed to initiate at least two cRPGRIP1 splice variants sharing the same 3′-terminal exon 19c. Transcription initiation sites were highly supported by conserved arrangements of cis-elements predicted in a bioinformatic analysis of orthologous RPGRIP1 promoter regions. Conclusions. The use of alternative transcription start and termination sites results in substantial heterogeneity of cRPGRIP1 transcripts, many of which are likely to have tissue-specific expression. The identified exon–intron structure of cRPGRIP1 isoforms provides a basis for evaluating the gene defects underlying inherited retinal disorders in dogs. PMID:21282582

  15. Expression profiles of NOS isoforms in gingiva of nNOS knockout mice.

    PubMed

    Ishioka, M; Ishizuka, Y; Shintani, S; Yanagisawa, T; Inoue, T; Sasaki, J; Watanabe, H

    2014-04-01

    Nitric oxide is a gaseous molecule associated with many distinct physiological functions, and is derived from L-arginine catalyzed by nitric oxide synthase (NOS). Nitric oxide synthase has 3 isoforms: nNOS, iNOS and eNOS. Although these NOS isoforms are believed to play an important role in gingival tissue, little information is available on their morphological dynamics. The aim of this study was to investigate the profiles of NOS isoforms in deficiency of nNOS in gingiva of mice. Twelve male (6 normal (C57BL/6) and 6 nNOS knockout) mice were used. All mice were 5-week-old, weighing approximately 20-25 g each. After sacrifice, the jaws of the mice were removed by mechanical means and specimens analyzed by histology, in situ hybridization and immunohistochemistry. Immunohistochemical observation revealed positive staining for iNOS and eNOS, especially in lamina propria. Similar results in the mRNA expression levels were shown by in situ hybridization analysis. It may suggest that iNOS and eNOS compensated nNOS deficiency in the gingiva of nNOS knockout mice.

  16. Differential isoform expression and protein localization from alternatively spliced Apetala2 in peanut under drought stress.

    PubMed

    Park, So-Yon; Grabau, Elizabeth

    2016-11-01

    APETALA2 (AP2) belongs to the AP2/Ethylene Responsive Factor (ERF) family and regulates expression levels of downstream stress responsive genes as a transcription factor. In this study, we cloned six different isoforms of AhAP2 from peanut (Arachis hypogaea). Four isoforms (AhAP2.1, AhAP2.2, AhAP2.3 and AhAP2.4) had both AP2/ERF DNA binding domains and ERF-associated amphiphilic repression (EAR) motifs. Two isoforms (AhAP2.5 and AhAP2.6) only had an EAR suppressor domain. After agroinfiltration, AhAP2.1, AhAP2.3, and AhAP2.4 fused to yellow fluorescent protein (YFP) showed localization to the nucleolus, which is the site of transcription and ribosome biogenesis. AhAP2.2-YFP showed a dispersed signal in the nucleus. AhAP2.5 and AhAP2.6 fused to YFP localized to both the nucleus and cytoplasm. In addition, increased levels of AhAP2.1 and AhAP2.2 transcripts were observed in drought-treated peanut leaves, suggesting differential transcriptional regulation under drought stress conditions.

  17. Cloning and identification of a novel thyroid hormone receptor β isoform expressed in the pituitary gland.

    PubMed

    Zhao, Rong-Lan; Sun, Bei; Liu, Ying; Li, Jing-Hua; Xiong, Wei-Li; Liang, Dong-Chun; Guo, Gang; Zuo, Ai-Jun; Zhang, Jing-Yu

    2014-04-01

    We have previously identified a novel Trβ isoform (TrβΔ) in the rat, in which a novel exon N (108 bps) was found between exon 3 and exon 4 of TrβΔ, which represents the only difference between TrβΔ and Trβ1. In this study, we searched for an elongated Trβ2-like subtype with one additional exon N. We successfully isolated the entire mRNA/cDNA of a novel elongated Trβ2 isoform via PCR in the rat pituitary gland. The mRNA/cDNA was only 108 bps (exon N) longer than that Trβ2, and the extension of the sequence was between exon 3 and 4 of Trβ. The whole sequence of this novel Trβ isoform has been published in NCBI GenBank (HM043807.1); it is named TRbeta2Delta (Trβ2Δ). In adult rat pituitary tissue, quantitative real-time RT-PCR analysis showed that the mRNA levels of Trβ2Δ and Trβ2 were roughly equal (P > 0.05). We cloned, expressed, and purified the His-Trβ2Δ protein [recombinant TRβ2Δ (rTRβ2Δ)]. SDS-PAGE and western blotting revealed that the molecular weight of rTRβ2Δ was 58.2 kDa. Using a radioligand binding assay and an electrophoretic mobility shift assay, rTRβ2Δ-bound T3 with high affinity and recognized thyroid hormone response element (TRE) binding sites. Finally, in vitro transfection experiments further confirmed that rTRβ2Δ binding T3 significantly promotes the transcription of target genes via the TRE. Here, we have provided evidence suggesting that rTRβ2Δ is a novel functional TR isoform.

  18. Cloning and expression of progesterone receptor isoforms A and B in bovine corpus luteum.

    PubMed

    Rekawiecki, Robert; Kowalik, Magdalena Karolina; Kotwica, Jan

    2015-09-01

    Progesterone (P4) affects a cell through its nuclear receptor (PGR), which has two main isoforms: A (PGRA) and B (PGRB). A partial section of previously unknown PGRB cDNA from cattle was cloned. Next, mRNA and protein levels for these two isoforms in corpora lutea (CL) collected during different stages of the oestrous cycle and pregnancy were determined. The PGRB mRNA level was highest on Days 2-5 of the oestrous cycle, decreased over the next few days (P<0.01) and increased again slightly on Days 17-20 (P<0.05). During pregnancy, PGRB mRNA was at its lowest level during Weeks 3-5 (P<0.01) and highest during Weeks 6-12 (P<0.01). The profile of PGRA mRNA levels was similar to that of PGRB throughout the oestrous cycle. The PGRA protein level was highest on Days 2-10 of the oestrous cycle, decreased continuously to its lowest concentration on Days 17-20 (P<0.01) and during Weeks 3-5 of pregnancy (P>0.05) and increased during Weeks 6-12 (P<0.05). PGRB protein concentration followed a similar pattern but at a markedly lower level. Both PGRA and PGRB isoforms are involved in the regulation of P4 action, especially in the newly formed CL and developed CL in the first trimester of pregnancy. These data suggest that the variable expression of these isoforms during the oestrous cycle may depend on the influence of P4.

  19. Single-Cell mRNA Profiling Reveals Cell-Type Specific Expression of Neurexin Isoforms

    PubMed Central

    Fuccillo, Marc V.; Földy, Csaba; Gökce, Özgün; Rothwell, Patrick E.; Sun, Gordon L.; Malenka, Robert C.; Südhof, Thomas C.

    2016-01-01

    Summary Neurexins are considered central organizers of synapse architecture that are implicated in neuropsychiatric disorders. Expression of neurexins in hundreds of alternatively spliced isoforms suggested that individual neurons might exhibit a cell type-specific neurexin expression pattern (a neurexin code). To test this hypothesis, we quantified the single-cell levels of neurexin isoforms and other trans-synaptic cell-adhesion molecules by microfluidics-based RT-PCR. We show that the neurexin repertoire displays pronounced cell-type specificity that is remarkably consistent within each type of neuron. Furthermore, we uncovered region-specific regulation of neurexin transcription and splice-site usage. Finally, we demonstrate that the transcriptional profiles of neurexins can be altered in an experience-dependent fashion by exposure to a drug of abuse. Our data provide evidence of cell type-specific expression patterns of multiple neurexins at the single-cell level, and suggest that expression of synaptic cell-adhesion molecules overlaps with other key features of cellular identity and diversity. PMID:26182417

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

    PubMed

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

    2016-11-01

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

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

    PubMed Central

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

    2016-01-01

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

  2. Expression of myosin isoforms in the smooth muscle of human corpus cavernosum.

    PubMed

    Koi, P T; Milhoua, P M; Monrose, V; Melman, A; DiSanto, M E

    2007-01-01

    The molecular interaction between smooth muscle (SM) myosin and actin in the corpus cavernosum (CC) determines the erectile state of the penis. A key mechanism regulating this interaction and subsequent development and maintenance of force is alternative splicing of SM myosin heavy chain (MHC) and 17 kDa essential SM myosin light chain (MLC) pre-mRNAs. Our aim was to examine the relative SM myosin isoform composition in human CC. Tissue samples were obtained from 18 patients with erectile dysfunction (ED), Peyronie's disease, or both. One specimen was obtained during a transgender operation. Patients then were stratified according to presence of diabetes mellitus, hypertension, ED, or Peyronie's disease, as well as failure of phosphodiesterase-5 (PDE5) inhibitors and history of previous pelvic or penile surgeries, radiation, or both. Our results revealed that all human CC samples expressed only the SM-A isoform. There was a predominance of SM2 isoform mRNA relative to SM1 across all samples, with a mean of 63.8%, which correlated with protein analysis by gel electrophoresis. A statistically significant difference was found between patients who had undergone previous pelvic surgery, radiation, or both and those who did not. The ratio of LC(17b) to LC(17a) was approximately 1:1 for all patients, with a mean of 48.9% LC(17b). Statistical difference was seen in the relative ratio of LC(17b) to LC(17a) among the group who failed conservative therapy with PDE5 inhibitors compared with all others. In conclusion, we determined the SM myosin isoform composition of human CC and present for the first time differences in relative myosin isoform expression among patients with several risk factors contributing to their cause of ED. Our data reflect the fact that alternative splicing events in the MHC and 17 kDa MLC pre-mRNA may be a possible molecular mechanism involved in the altered contractility of the CCSM in patients with ED.

  3. A new animal model for modulating myosin isoform expression by altered mechanical activity

    NASA Technical Reports Server (NTRS)

    Caiozzo, V. J.; Ma, E.; McCue, S. A.; Smith, E.; Herrick, R. E.; Baldwin, K. M.

    1992-01-01

    The purpose of this study was to develop a new rodent model that is capable of delineating the importance of mechanical loading on myosin heavy chain (MHC) isoform expression of the plantar and dorsi flexor muscles of the ankle. The essential components of this system include 1) stimulating electrodes that are chronically implanted into a muscle, allowing for the control of the activation pattern of the target muscle(s); 2) a training apparatus that translates the moment of the ankle into a linear force; and 3) a computer-controlled Cambridge 310 ergometer. The isovelocity profile of the ergometer ensured that the medial gastrocnemius (MG) produced forces that were > 90% of maximal isometric force (Po), and the eccentric contractions of the tibialis anterior (TA) were typically 120% of Po. Both the concentric and eccentric training programs produced statistically significant increases in the muscle mass of the MG (approximately 15%) and TA (approximately 7%) as well as a decrease in myofibrillar adenosinetriphosphatase activity. Both the white and red regions of the MG and TA exhibited significant increases in the relative content of the type IIa MHC and concomitant decreases in type IIb MHC expression. Although the red regions of the MG and red TA contained approximately 10% type I MHC, the training programs did not affect this isoform. It appears that when a fast-twitch muscle is stimulated at a high frequency (100 Hz) and required to contract either concentrically or eccentrically under high loading conditions, the expression of the type IIa MHC isoform will be upregulated, whereas that of the type IIb MHC will be concomitantly downregulated.

  4. Dietary Macronutrient Composition Directs ChREBP Isoform Expression and Glucose Metabolism in Mice

    PubMed Central

    Howard, Victor; Youngs, Kristina; Cowley, Michael A.; Sleeman, Mark W.

    2016-01-01

    Carbohydrate response element binding protein (ChREBP) is a lipogenic transcription factor that is thought to be involved in the development of hepatic steatosis and insulin resistance. Increased ChREBP expression in liver results in increased hepatic steatosis, and the isoform ChREBPβ in adipose tissue can predict insulin sensitivity in obese humans. As ChREBP is activated by glucose, it was postulated that the composition of diet would regulate ChREBP isoform expression in metabolically relevant tissues. We compared the effects of diets with high complex carbohydrate, high fat, or a normal chow on ChREBP expression and metabolic parameters in C57BL/6 mice. We found that diets high in fat decrease ChREBP expression in adipose tissue, but isocaloric diets high in carbohydrate have no effect. Interestingly, this decrease in adipose ChREBP was associated with increased inflammatory markers. In the same animals a high carbohydrate diet induced a robust increase in hepatic ChREBPβ expression (≈2-fold; p = 0.0002), but little detectable change in the more abundant ChREBPα transcript. This change was accompanied by increased expression of target genes liver pyruvate kinase (p<0.0001), acetyl-CoA carboxylase (p = 0.0191) and stearoyl-CoA desaturase-1 (p = 0.0045). This increase in ChREBP expression was associated with increased hepatic steatosis, despite no changes in body weight or body fat when compared to chow-fed mice. Unexpectedly, mice fed a high carbohydrate diet displayed enhanced sensitivity to exogenous insulin, despite having mild glucose intolerance and increased liver steatosis. In summary, we have shown the composition of diet can selectively regulate ChREBP isoform expression in a tissue specific manner. Furthermore, we have shown a high complex carbohydrate diet selectively increases hepatic ChREBPβ expression, which associates with hepatic steatosis but not insulin resistance. In contrast, a high fat diet reduces adipose ChREBP, which associates

  5. IDP-ASE: haplotyping and quantifying allele-specific expression at the gene and gene isoform level by hybrid sequencing.

    PubMed

    Deonovic, Benjamin; Wang, Yunhao; Weirather, Jason; Wang, Xiu-Jie; Au, Kin Fai

    2016-11-28

    Allele-specific expression (ASE) is a fundamental problem in studying gene regulation and diploid transcriptome profiles, with two key challenges: (i) haplotyping and (ii) estimation of ASE at the gene isoform level. Existing ASE analysis methods are limited by a dependence on haplotyping from laborious experiments or extra genome/family trio data. In addition, there is a lack of methods for gene isoform level ASE analysis. We developed a tool, IDP-ASE, for full ASE analysis. By innovative integration of Third Generation Sequencing (TGS) long reads with Second Generation Sequencing (SGS) short reads, the accuracy of haplotyping and ASE quantification at the gene and gene isoform level was greatly improved as demonstrated by the gold standard data GM12878 data and semi-simulation data. In addition to methodology development, applications of IDP-ASE to human embryonic stem cells and breast cancer cells indicate that the imbalance of ASE and non-uniformity of gene isoform ASE is widespread, including tumorigenesis relevant genes and pluripotency markers. These results show that gene isoform expression and allele-specific expression cooperate to provide high diversity and complexity of gene regulation and expression, highlighting the importance of studying ASE at the gene isoform level. Our study provides a robust bioinformatics solution to understand ASE using RNA sequencing data only.

  6. Pulmonary expression of nitric oxide synthase isoforms in sheep with smoke inhalation and burn injury.

    PubMed

    Cox, Robert A; Jacob, Sam; Oliveras, Gloria; Murakami, Kazunori; Enkhbaatar, Perenlei; Traber, Lillian; Schmalstieg, Frank C; Herndon, David N; Traber, Daniel L; Hawkins, Hal K

    2009-03-01

    Previous studies have indicated increased plasma levels of inducible nitric oxide synthase in lung. This study further examines the pulmonary expression of nitric oxide synthase (NOS) isoforms in an ovine model of acute lung injury induced by smoke inhalation and burn injury (S+B injury). Female range bred sheep (4 per group) were sacrificed at 4, 8, 12, 24, and 48 hours after injury and immunohistochemistry was performed in tissues for various NOS isoforms. The study indicates that in uninjured sheep lung, endothelial (eNOS) is constitutively expressed in the endothelial cells associated with the airways and parenchyma, and in macrophages. Similarly, neuronal (nNOS) is constitutively present in the mucous cells of the epithelium and in neurons of airway ganglia. In uninjured lung, inducible (iNOS) was present in bronchial secretory cells and macrophages. In tissue after S+B injury, new expression of iNOS was evident in bronchial ciliated cells, basal cells, and mucus gland cells. In the parenchyma, a slight increase in iNOS immunostaining was seen in type I cells at 12 and 24 hours after injury only. Virtually no change in eNOS or nNOS was seen after injury.

  7. Smad2 isoforms are differentially expressed during mouse brain development and aging.

    PubMed

    Ueberham, Uwe; Lange, Peggy; Ueberham, Elke; Brückner, Martina K; Hartlage-Rübsamen, Maike; Pannicke, Thomas; Rohn, Susanne; Cross, Michael; Arendt, Thomas

    2009-08-01

    Smad2 and Smad3 are central molecules of the TGFbeta and activin receptor complex mediated intracellular signaling pathway. They function as important transcription factors playing essential roles in brain development. Interestingly they are also known to be involved in the pathogenesis of various neurological disorders (including Alzheimer's disease). Due to structural differences in the N-terminal Mad homology domain 1, Smad2 and Smad3 differ in their ability to bind DNA directly. A splice form of Smad2 lacking exon3, Smad2(Deltaexon3), assumes features of Smad3, in that it can directly bind to DNA resulting in a functional hybrid of Smad2 and Smad3 properties. There is very little information available on the expression of Smad2 isoforms in the brain. We report here that Smad2(Deltaexon3) is the most abundant of the two Smad2 isoforms in mouse brain and that Smad expression pattern alters during development and aging. Neuronal expression of Smad2(Deltaexon3) was confirmed by a single-cell PCR approach. Moreover, Smad2(Deltaexon3) predominates in the nuclear fraction of neurons, suggesting special function during brain differentiation. Our data indicate that there may be a specific role for Smad2(Deltaexon3) in neurons.

  8. Molecular cloning, genomic organization, and expression of a testicular isoform of hormone-sensitive lipase

    SciTech Connect

    Holst, L.S.; Laurell, H.; Holm, C.

    1996-08-01

    By catalyzing the rate-limiting step in adipose tissue lipolysis, hormone-sensitive lipase (HSL) is an important regulator of energy homeostasis. The role and importance of HSL in tissues other than adipose are poorly understood. We report here the cloning and expression of a testicular isoform, designated HSL{sub tes}. Due to an addition of amino acids at the NH{sub 2}-termini, rat and human HSL{sub tes} consist of 1068 and 1076 amino acids, respectively, compared to the 768 and 775 amino acids, respectively, of the adipocyte isoform (HSL{sub adi}). A novel exon of 1.2 kb, encoding the human testis-specific amino acids, was isolated and mapped to the HSL gene, 16 kb upstream of the exons encoding HSL{sub adi}. The transcribed mRNA of 3.9 kb was specifically expressed in testis. No significant similarity with other known proteins was found for the testis-specific sequence. The amino acid composition differs from the HSL{sub adi} sequence, with a notable hydrophilic character and a high content of prolines and glutamines. COS cells, transfected by the 3.9-kb human testis cDNA, expressed a protein of the expected molecular mass (M{sub r} {approximately}120,000) that exhibited catalytic activity similar to that of HSL{sub adi}. Immunocytochemistry localized HSL to elongating spermatids and spermatozoa; HSL was not detected in interstitial cells. 34 refs., 5 figs.

  9. Differential expression of laminin isoforms in diabetic nephropathy and other renal diseases.

    PubMed

    Setty, Suman; Michael, Alfred A; Fish, Alfred J; Michael Mauer, S; Butkowski, Ralph J; Virtanen, Ismo; Kim, Youngki

    2012-06-01

    Laminin a non-collagenous glycoprotein is a major component of the renal glomerular basement membrane and mesangium. Thus far eleven distinct chains have been described, permutations of which make up 15 laminin isoforms. Laminin molecules interact with cells and other matrix molecules during organ development and differentiation. We studied the distribution of laminin isoforms in patients with type 1 diabetic nephropathy, membranous nephropathy, membranoproliferative glomerulonephritis and IgA nephropathy/ Henoch-Schönlein purpura. Immunofluorescence microscopic studies with laminin-chain-specific antibodies to the α1, α2, α5, β1, β2 and γ1 chains detected α2, β1 and γ1 chain expression in the normal mesangium and α5, β2 and γ1 in normal glomerular basement membrane. Significantly, constituents of the glomerular basement membrane, α5, β2 and γ1 chains were overexpressed in kidneys with diabetic nephropathy. Initially the constituents of the mesangium increased commensurate with the degree of mesangial expansion and degree of diabetic nephropathy. Reduction in α2 chain intensity was observed with severe mesangial expansion and in the areas of nodular glomerulosclerosis. In addition, with late disease aberrant expression of α2 and β2 chains was observed in the mesangium. Glomerular basement membrane in renal disease overexpressed molecules normally present in that location. In summary, the alterations in basement membrane composition in various renal diseases seem to not only reflect the balance between synthesis and degradation of normal basement membrane constituents, but also their aberrant expression.

  10. Expression of eight distinct MHC isoforms in bovine striated muscles: evidence for MHC-2B presence only in extraocular muscles.

    PubMed

    Toniolo, L; Maccatrozzo, L; Patruno, M; Caliaro, F; Mascarello, F; Reggiani, C

    2005-11-01

    This study aimed to analyse the expression of myosin heavy chain (MHC) isoforms in bovine muscles, with particular attention to the MHC-2B gene. Diaphragm, longissimus dorsi, masseter, several laryngeal muscles and two extraocular muscles (rectus lateralis and retractor bulbi) were sampled in adult male Bos taurus (age 18-24 months, mass 400-500 kg) and analysed by RT-PCR, gel electrophoresis and immunohistochemistry. Transcripts and proteins corresponding to eight MHC isoforms were identified: MHC-alpha and MHC-beta/slow (or MHC-1), two developmental isoforms (MHC-embryonic and MHC-neonatal), three adult fast isoforms (MHC-2A, MHC-2X and MHC-2B) and the extraocular isoform MHC-Eo. All eight MHC isoforms were found to be co-expressed in extrinsic eye muscles, retractor bulbi and rectus lateralis, four (beta/slow, 2A, 2X, neonatal) in laryngeal muscles, three (beta/slow, 2A and 2X) in trunk and limb muscles and two (beta/slow and alpha) in masseter. The expression of MHC-2B and MHC-Eo was restricted to extraocular muscles. Developmental MHC isoforms (neonatal and embryonic) were only found in specialized muscles in the larynx and in the eye. MHC-alpha was only found in extraocular and masseter muscle. Single fibres dissected from masseter, diaphragm and longissimus were classified into five groups (expressing, respectively, beta/slow, alpha, slow and 2A, 2A and 2X) on the basis of MHC isoform electrophoretical separation, and their contractile properties [maximum shortening velocity (v(0)) and isometric tension (P(0))] were determined. v(0) increased progressively from slow to fast 2A and fast 2X, whereas hybrid 1-2A fibres and fibres containing MHC-alpha were intermediate between slow and fast 2A.

  11. EP3 Receptor Isoforms are Differentially Expressed in Subpopulations of Primate Granulosa Cells and Couple to Unique G-Proteins

    PubMed Central

    Kim, Soon Ok; Dozier, Brandy L.; Kerry, Julie A.; Duffy, Diane M.

    2013-01-01

    Prostaglandin E2 produced within the ovarian follicle is necessary for ovulation. Prostaglandin E2 is recognized by four distinct G-protein coupled receptors. Among them, PTGER3 (also known as EP3) is unique in that mRNA splicing generates multiple isoforms. Each isoform has a distinct amino acid composition in the C-terminal region, which is involved in G-protein coupling. To determine if monkey EP3 isoforms couple to different G-proteins, each EP3 isoform was expressed in Chinese hamster ovary (CHO) cells, and intracellular signals were examined after stimulation with the EP3 agonist sulprostone. Stimulation of EP3 isoform 5 (EP3-5) reduced cyclic adenosine monophosphate (cAMP) in a pertussis toxin-sensitive manner, indicating involvement of Gαi. Stimulation of EP3-9 increased cAMP, which was reduced by the general G-protein inhibitor GDP-β-S, and also increased intracellular calcium, which was reduced by pertussis toxin and GDP-β-S. So, EP3-9 likely couples to both Gαs and a pertussis toxin-sensitive G-protein to regulate intracellular signals. Stimulation of EP3-14 increased cAMP, which was further increased by pertussis toxin, so EP3-14 likely regulates cAMP via multiple G-proteins. Granulosa cell expression of all EP3 isoforms increased in response to an ovulatory dose of hCG. Two EP3 isoforms were differentially expressed in functional subpopulations of granulosa cells. EP3-5 was low in granulosa cells at the follicle apex while EP3-9 was high in cumulus granulosa cells. Differential expression of EP3 isoforms may yield different intracellular responses to prostaglandin E2 in granulosa cell subpopulations, contributing to the different roles played by granulosa cell subpopulations in the process of ovulation. PMID:24062570

  12. Isoform-specific expression and ratio changes accompany oxidant-induced peripherin aggregation in a neuroblastoma cell line.

    PubMed

    McLean, Jesse R; Robertson, Janice

    2011-11-08

    The type III intermediate filament peripherin is found associated with pathological inclusions present within motor neurons of patients with amyotrophic lateral sclerosis (ALS). Peripherin intra-isoform associations contribute to filament network formation at defined stoichiometric ratios. Distinct biochemical signatures characterize peripherin isoform expression in traumatic neuronal injury and motor neuron disease, while disruptions to peripherin alternative splicing or translation are associated with inclusion formation. In our efforts to identify pathological relationships between peripherin isoform expression and inclusion formation, we provide evidence of peripherin isoform-specific expression and ratio changes with concomitant, dose-dependent inclusion formation in response to oxidative stress. Upon increasing exposure to physiologically relevant levels of hydrogen peroxide in Neuro-2a cells, we observed a significant increase and decrease in peripherin isoforms Per-58 and Per-45, respectively, with peripherin-specific perikaryal aggregation of filaments 10-15 μm in diameter. Interestingly, peripherin-immunoreactive inclusions showed no overt carbonylation, suggesting that aggregation may serve a physiologically relevant role during oxidative stress. These findings provide novel insight into the biological significance of peripherin isoforms and inclusion formation, with relevance to the pathology of ALS.

  13. Cell-Specific Fine-Tuning of Neuronal Excitability by Differential Expression of Modulator Protein Isoforms

    PubMed Central

    Jepson, James; Sheldon, Amanda; Shahidullah, Mohammad; Fei, Hong; Koh, Kyunghee

    2013-01-01

    SLOB (SLOWPOKE-binding protein) modulates the Drosophila SLOWPOKE calcium-activated potassium channel. We have shown previously that SLOB deletion or RNAi knockdown decreases excitability of neurosecretory pars intercerebralis (PI) neurons in the adult Drosophila brain. In contrast, we found that SLOB deletion/knockdown enhances neurotransmitter release from motor neurons at the fly larval neuromuscular junction, suggesting an increase in excitability. Because two prominent SLOB isoforms, SLOB57 and SLOB71, modulate SLOWPOKE channels in opposite directions in vitro, we investigated whether divergent expression patterns of these two isoforms might underlie the differential modulation of excitability in PI and motor neurons. By performing detailed in vitro and in vivo analysis, we found strikingly different modes of regulatory control by the slob57 and slob71 promoters. The slob71, but not slob57, promoter contains binding sites for the Hunchback and Mirror transcriptional repressors. Furthermore, several core promoter elements that are absent in the slob57 promoter coordinately drive robust expression of a luciferase vector by the slob71 promoter in vitro. In addition, we visualized the expression patterns of the slob57 and slob71 promoters in vivo and found clear spatiotemporal differences in promoter activity. SLOB57 is expressed prominently in adult PI neurons, whereas larval motor neurons exclusively express SLOB71. In contrast, at the larval neuromuscular junction, SLOB57 expression appears to be restricted mainly to a subset of glial cells. Our results illustrate how the use of alternative transcriptional start sites within an ion channel modulator locus coupled with functionally relevant alternative splicing can be used to fine-tune neuronal excitability in a cell-specific manner. PMID:24133277

  14. Expression of CD150 in tumors of the central nervous system: identification of a novel isoform.

    PubMed

    Romanets-Korbut, Olga; Najakshin, Alexander M; Yurchenko, Mariya; Malysheva, Tatyana A; Kovalevska, Larysa; Shlapatska, Larysa M; Zozulya, Yuriy A; Taranin, Alexander V; Horvat, Branka; Sidorenko, Svetlana P

    2015-01-01

    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.

  15. The distribution and expression of the two isoforms of DNA topoisomerase II in normal and neoplastic human tissues.

    PubMed Central

    Turley, H.; Comley, M.; Houlbrook, S.; Nozaki, N.; Kikuchi, A.; Hickson, I. D.; Gatter, K.; Harris, A. L.

    1997-01-01

    In mammalian cells, there are two isoforms of DNA topoisomerase II, designated alpha (170-kDa form) and beta (180-kDa form). Previous studies using cell lines have shown that the topoisomerase IIalpha and beta isoforms are differentially regulated during the cell cycle and in response to changes in growth state. Moreover, both isoforms can act as targets for a range of anti-tumour drugs. Here, we have analysed the normal tissue distribution in humans of topoisomerase IIalpha and beta using isoform-specific antibodies. In addition, we have studied expression of these isoforms in 69 primary tumour biopsies, representative either of tumours that are responsive to topoisomerase II-targeting drugs (breast, lung, lymphoma and seminoma) or of those that show de novo drug resistance (colon). Topoisomerase IIalpha was expressed exclusively in the proliferating compartments of all normal tissues, and was detectable in both the cell nucleus and cytoplasm. In biologically aggressive or rapidly proliferating tumours (e.g. high-grade lymphomas and seminomas), there was a high level of topoisomerase IIalpha, although expression was still detectable in colon tumours, indicating that expression of this isoform is not sufficient to explain the intrinsic drug resistance of colon tumours. Topoisomerase IIbeta was expressed ubiquitously in vivo and was localized in both the nucleoli and the nucleoplasm. This isoform was present in quiescent cell populations, but was expressed at a generally higher level in all tumours and proliferating cells than in normal quiescent tissues. We conclude that topoisomerase IIalpha is a strict proliferation marker in normal and neoplastic cells in vivo, but that topoisomerase IIbeta has a much more general cell and tissue distribution than has topoisomerase IIalpha. The apparent up-regulation of topoisomerase IIbeta in neoplastic cells has implications for the response of patients to anti-tumour therapies that include topoisomerase II-targeting drugs

  16. A truncated, activin-induced Smad3 isoform acts as a transcriptional repressor of FSHβ expression in mouse pituitary.

    PubMed

    Kim, So-Youn; Zhu, Jie; Woodruff, Teresa K

    2011-08-06

    The receptor-regulated protein Smad3 is key player in the signaling cascade stimulated by the binding of activin to its cell surface receptor. Upon phosphorylation, Smad3 forms a heterocomplex with Smad2 and Smad4, translocates to the nucleus and acts as a transcriptional co-activator. We have identified a unique isoform of Smad3 that is expressed in mature pituitary gonadotropes. 5' RACE revealed that this truncated Smad3 isoform is transcribed from an ATG site within exon 4 and consists of 7 exons encoding half of the linker region and the MH2 region. In pituitary cells, the truncated Smad3 isoform was phosphorylated upon activin treatment, in a manner that was temporally distinct from the phosphorylation of full-length Smad3. Activin-induced phosphorylation of Smad3 and the truncated Smad3 isoform was blocked by both follistatin and siRNA-mediated knockdown of Smad3. The truncated Smad3 isoform antagonized Smad3-mediated, activin-responsive promoter activity. We propose that the pituitary gonadotrope contains an ultra-short, activin-responsive feedback loop utilizing two different isoforms of Smad3, one which acts as an agonist (Smad3) and another that acts as an intracrine antagonist (truncated Smad3 isoform) to regulate FSHβ production.

  17. Expression of pro- and anti-angiogenic isoforms of VEGF is differentially regulated by splicing and growth factors

    PubMed Central

    Nowak, Dawid G.; Woolard, Jeanette; Amin, Elianna Mohamed; Konopatskaya, Olga; Saleem, Moin A.; Churchill, Amanda J.; Ladomery, Michael R.; Harper, Steven J.; Bates, David O.

    2008-01-01

    Summary Vascular endothelial growth factor A (VEGFA; hereafter referred to as VEGF) is a key regulator of physiological and pathological angiogenesis. Two families of VEGF isoforms are generated by alternate splice-site selection in the terminal exon. Proximal splice-site selection (PSS) in exon 8 results in pro-angiogenic VEGFxxx isoforms (xxx is the number of amino acids), whereas distal splice-site selection (DSS) results in anti-angiogenic VEGFxxxb isoforms. To investigate control of PSS and DSS, we investigated the regulation of isoform expression by extracellular growth factor administration and intracellular splicing factors. In primary epithelial cells VEGFxxxb formed the majority of VEGF isoforms (74%). IGF1, and TNFα treatment favoured PSS (increasing VEGFxxx) whereas TGFβ1 favoured DSS, increasing VEGFxxxb levels. TGFβ1 induced DSS selection was prevented by inhibition of p38 MAPK and the Clk/sty (CDC-like kinase, CLK1) splicing factor kinase family, but not ERK1/2. Clk phosphorylates SR protein splicing factors ASF/SF2, SRp40 and SRp55. To determine whether SR splicing factors alter VEGF splicing, they were overexpressed in epithelial cells, and VEGF isoform production assessed. ASF/SF2, and SRp40 both favoured PSS, whereas SRp55 upregulated VEGFxxxb (DSS) isoforms relative to VEGFxxx. SRp55 knockdown reduced expression of VEGF165b. Moreover, SRp55 bound to a 35 nucleotide region of the 3′UTR immediately downstream of the stop codon in exon 8b. These results identify regulation of splicing by growth and splice factors as a key event in determining the relative pro- versus anti-angiogenic expression of VEGF isoforms, and suggest that p38 MAPK-Clk/sty kinases are responsible for the TGFβ1-induced DSS selection, and identify SRp55 as a key regulatory splice factor. PMID:18843117

  18. CD44v3 and v6 variant isoform expression correlates with poor prognosis in early-stage vulvar cancer.

    PubMed Central

    Tempfer, C.; Sliutz, G.; Haeusler, G.; Speiser, P.; Reinthaller, A.; Breitenecker, G.; Vavra, N.; Kainz, C.

    1998-01-01

    Expression of alternatively spliced CD44 isoforms has been reported to correlate with poor prognosis in human squamous cell cancers, i.e. squamous cell cancer of the lung and cervix. The aim of this study was to evaluate whether CD44 isoform expression is a prognostic factor in early-stage squamous cell cancer of the vulva. Seventy cases of squamous cell carcinoma of the vulva International Federation of Gynaecology and Obstetrics (FIGO) stage I were examined immunohistochemically for expression of CD44 isoforms. We used four different variant exon sequence-specific murine monoclonal antibodies to epitopes encoded by exons v3, v5, v6 and v7-8 of human variant CD44. The correlation of CD44 expression with histological grade and disease-free and overall survival was investigated. CD44 isoforms CD44v3, CD44v5, CD44v6 and CD44v7-8 were detected in 28% (20/70), 47% (33/70), 33% (23/70) and 17% (12/70) of the tumour samples respectively. Patients suffering from tumours expressing CD44v6 had a poorer relapse-free (log-rank test, P = 0.02) and overall survival (log-rank test, P = 0.03). Likewise, patients suffering from tumours expressing CD44v3 had a poorer relapse-free (log-rank test, P = 0.04) and overall survival (log-rank test, P = 0.01). Expression of CD44v5 and CD44v7-8 did not compromise the patients' outcome. Histological grade did not correlate with CD44 isoform expression. Immunohistochemically detected expression of CD44 isoforms containing variant exon v6 or v3 is correlated with a poor relapse-free and overall survival in FIGO stage I vulvar cancer patients. PMID:9792156

  19. Expression of ESE-3 Isoforms in Immunogenic and Tolerogenic Human Monocyte-Derived Dendritic Cells

    PubMed Central

    Sprater, Florian; Hovden, Arnt-Ove; Appel, Silke

    2012-01-01

    Dendritic cells (DC) are the only hematopoietic cells expressing the epithelial specific Ets transcription factor ESE-3. Here we analyzed presence and quantity of isoforms ESE-3a, ESE-3b and ESE-3j in various immunogenic and tolerogenic human monocyte-derived DC (moDC) and blood DC populations using quantitative real time PCR and immunoblot analyses. ESE-3a and ESE-3b were detectable in all moDC populations with ESE-3b being the main transcript. ESE-3b expression was upregulated in immunogenic moDC and downregulated in tolerogenic moDC compared to immature moDC. ESE-3a had similar transcript levels in immature and immunogenic moDC and had very low levels in tolerogenic moDC. In blood DC populations only splice variant ESE-3b was detectable. ESE-3j was not detectable in any of the DC populations. These findings suggest that ESE-3b is the functionally most important ESE-3 isoform in DC. PMID:23185370

  20. Expression of ESE-3 isoforms in immunogenic and tolerogenic human monocyte-derived dendritic cells.

    PubMed

    Sprater, Florian; Hovden, Arnt-Ove; Appel, Silke

    2012-01-01

    Dendritic cells (DC) are the only hematopoietic cells expressing the epithelial specific Ets transcription factor ESE-3. Here we analyzed presence and quantity of isoforms ESE-3a, ESE-3b and ESE-3j in various immunogenic and tolerogenic human monocyte-derived DC (moDC) and blood DC populations using quantitative real time PCR and immunoblot analyses. ESE-3a and ESE-3b were detectable in all moDC populations with ESE-3b being the main transcript. ESE-3b expression was upregulated in immunogenic moDC and downregulated in tolerogenic moDC compared to immature moDC. ESE-3a had similar transcript levels in immature and immunogenic moDC and had very low levels in tolerogenic moDC. In blood DC populations only splice variant ESE-3b was detectable. ESE-3j was not detectable in any of the DC populations. These findings suggest that ESE-3b is the functionally most important ESE-3 isoform in DC.

  1. Neurexin 1 (NRXN1) Splice Isoform Expression During Human Neocortical Development and Aging

    PubMed Central

    Jenkins, Aaron K.; Paterson, Clare; Wang, Yanhong; Hyde, Thomas M.; Kleinman, Joel E.; Law, Amanda J.

    2015-01-01

    Neurexin 1 (NRXN1), a presynaptic adhesion molecule, is implicated in several neurodevelopmental disorders characterized by synaptic dysfunction including, autism, intellectual disability, and schizophrenia. To gain insight into NRXN1’s involvement in human cortical development we used quantitative real time PCR to examine the expression trajectories of NRXN1, and its predominant isoforms NRXN1-α and NRXN1-β in prefrontal cortex from fetal stages to aging. Additionally, we investigated whether prefrontal cortical expression levels of NRXN1 transcripts are altered in schizophrenia or bipolar disorder in comparison to non-psychiatric control subjects. We observed that all three NRXN1 transcripts were highly expressed during human fetal cortical development, dramatically increasing with gestational age. In the postnatal DLPFC, expression levels were negatively correlated with age, peaking at birth until approximately 3 years of age, after which levels declined dramatically to be stable across the lifespan. NRXN1-β expression was modestly but significantly elevated in the brains of patients with schizophrenia compared to non-psychiatric controls, whereas NRXN1-α expression was increased in bipolar disorder. These data provide novel evidence that NRXN1 expression is highest in human prefrontal cortex during critical developmental windows relevant to the onset and diagnosis of a range of neurodevelopmental disorders, and that NRXN1 expression may be differentially altered in neuropsychiatric disorders. PMID:26216298

  2. Discoidin domain receptor 1: isoform expression and potential functions in cirrhotic human liver.

    PubMed

    Song, Sunmi; Shackel, Nicholas A; Wang, Xin M; Ajami, Katerina; McCaughan, Geoffrey W; Gorrell, Mark D

    2011-03-01

    Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that binds and is activated by collagens. Transcriptional profiling of cirrhosis in human liver using a DNA array and quantitative PCR detected elevated mRNA expression of DDR1 compared with that in nondiseased liver. The present study characterized DDR1 expression in cirrhotic and nondiseased human liver and examined the cellular effects of DDR1 expression. mRNA expression of all five isoforms of DDR1 was detected in human liver, whereas DDR1a demonstrated differential expression in liver with hepatitis C virus and primary biliary cirrhosis compared with nondiseased liver. In addition, immunoblot analysis detected shed fragments of DDR1 more readily in cirrhotic liver than in nondiseased liver. Inasmuch as DDR1 is subject to protease-mediated cleavage after prolonged interaction with collagen, this differential expression may indicate more intense activation of DDR1 protein in cirrhotic compared with nondiseased liver. In situ hybridization and immunofluorescence localized intense DDR1 mRNA and protein expression to epithelial cells including hepatocytes at the portal-parenchymal interface and the luminal aspect of the biliary epithelium. Overexpression of DDR1a altered hepatocyte behavior including increased adhesion and less migration on extracelular matrix substrates. DDR1a regulated extracellular expression of matrix metalloproteinases 1 and 2. These data elucidate DDR1 function pertinent to cirrhosis and indicate the importance of epithelial cell-collagen interactions in chronic liver injury.

  3. Monoclonal antibodies against muscle actin isoforms: epitope identification and analysis of isoform expression by immunoblot and immunostaining in normal and regenerating skeletal muscle

    PubMed Central

    Chaponnier, Christine; Gabbiani, Giulio

    2016-01-01

    Higher vertebrates (mammals and birds) express six different highly conserved actin isoforms that can be classified in three subgroups: 1) sarcomeric actins, α-skeletal (α-SKA) and α-cardiac (α-CAA), 2) smooth muscle actins (SMAs), α-SMA and γ-SMA, and 3) cytoplasmic actins (CYAs), β-CYA and γ-CYA. The variations among isoactins, in each subgroup, are due to 3-4 amino acid differences located in their acetylated N-decapeptide sequence. The first monoclonal antibody (mAb) against an actin isoform (α-SMA) was produced and characterized in our laboratory in 1986 (Skalli  et al., 1986) . We have further obtained mAbs against the 5 other isoforms. In this report, we focus on the mAbs anti-α-SKA and anti-α-CAA obtained after immunization of mice with the respective acetylated N-terminal decapeptides using the Repetitive Immunizations at Multiple Sites Strategy (RIMMS). In addition to the identification of their epitope by immunoblotting, we describe the expression of the 2 sarcomeric actins in mature skeletal muscle and during muscle repair after micro-lesions. In particular, we analyze the expression of α-CAA, α-SKA and α-SMA by co-immunostaining in a time course frame during the muscle repair process. Our results indicate that a restricted myocyte population expresses α-CAA and suggest a high capacity of self-regeneration in muscle cells. These antibodies may represent a helpful tool for the follow-up of muscle regeneration and pathological changes. PMID:27335638

  4. Divergent tropism of HHV-6AGS and HHV-6BPL1 in T cells expressing different CD46 isoform patterns.

    PubMed

    Hansen, Aida S; Bundgaard, Bettina B; Biltoft, Mette; Rossen, Litten S; Höllsberg, Per

    2017-02-01

    CD46 is a receptor for HHV-6A, but its role as a receptor for HHV-6B is controversial. The significance of CD46 isoforms for HHV-6A and HHV-6B tropism is unknown. HHV-6AGS was able to initiate transcription of the viral genes U7 and U23 in the CD46(+)CD134(-) T-cell lines Peer, Jurkat, Molt3, and SupT1, whereas HHV-6BPL1 was only able to do so in Molt3 and SupT1, which expressed a CD46 isoform pattern different from Peer and Jurkat. The HHV-6BPL1-susceptible T-cell lines were characterized by low expression of the CD46 isoform BC2 and domination of isoforms containing the cytoplasmic tail, CYT-1. A HHV-6BPL1 susceptible cell line, Be13, changed over time its CD46 isoform pattern to resemble Peer and Jurkat and concomitantly lost its susceptibility to HHV-6BPL1 but not HHV-6AGS infection. We propose that isoforms of CD46 impact on HHV-6B infection and thereby in part explain the distinct tropism of HHV-6AGS and HHV-6BPL1.

  5. Daily Variations in the Glycerol-3-Phosphate Dehydrogenase Isoforms Expression in Triatoma infestans Flight Muscles

    PubMed Central

    Stroppa, María M.; Carriazo, Carlota S.; Gerez de Burgos, Nelia M.; Garcia, Beatríz A.

    2014-01-01

    Triatoma infestans, the main vector of Chagas disease, is a blood-sucking insect. Flight dispersal of adults is the most important mechanism for reinfestation of houses after insecticide spraying. Flight muscles have two glycerol-3-phosphate dehydrogenase (GPDH) isoforms: GPDH-1 is involved in flight metabolism and GPDH-2 provides lipid precursors. In this study, we explored the profile of GPDH expression in females and males adult flight muscles under light/dark cycle, constant light, and constant dark conditions. Under constant dark conditions, GPDH-1 flight muscles of T. infestans showed a rhythmic pattern of transcription synchronous with a rhythmic profile of activity suggesting regulation by the endogenous circadian clock. Otherwise, the GPDH-2 expression analysis showed no regulation by the endogenous clock, but showed that an external factor, such as the dark/light period, was necessary for synchronization of GPDH-2 transcription and activity. PMID:24914000

  6. Differential effects of heme oxygenase isoforms on heme mediation of endothelial intracellular adhesion molecule 1 expression.

    PubMed

    Wagener, F A; da Silva, J L; Farley, T; de Witte, T; Kappas, A; Abraham, N G

    1999-10-01

    Heme oxygenase (HO), by catabolizing heme to bile pigments, down-regulates cellular hemoprotein, hemoglobin, and heme; the latter generates pro-oxidant products, including free radicals. Two HO isozymes, the products of distinct genes, have been described; HO-1 is the inducible isoform, whereas HO-2 is suggested to be constitutively expressed. We studied the inducing effect of several metal compounds (CoCl(2), stannic mesoporphyrin, and heme) on HO activity. Additionally, we studied HO-1 expression in experimental models of adhesion molecule expression produced by heme in endothelial cells, and the relationship of HO-1 expression to the induced adhesion molecules. Flow cytometry analysis showed that heme induces intracellular adhesion molecule 1 (ICAM-1) expression in a concentration (10-100 microM)- and time (1-24 h)-dependent fashion in human umbilical vein endothelial cells. Pretreatment with stannic mesoporphyrin, an inhibitor of HO activity, caused a 2-fold increase in heme-induced ICAM-1 expression. In contrast, HO induction by CoCl(2) decreased heme-induced ICAM-1 expression by 33%. To examine the contribution of HO-1 and HO-2 to endothelial HO activity, specific antisense oligonucleotides (ODNs) of each isoform were tested for their specificity to inhibit HO activity in cells exposed to heme. Endothelial cells exposed to heme elicited increased HO activity, which was prevented (70%) by HO-1 antisense ODNs. HO-2 antisense ODN inhibited heme-induced HO activity by 21%. Addition of HO-1 antisense ODNs prevented heme degradation and resulted in elevation of microsomal heme. Western blot analysis showed that HO-1 antisense ODNs selectively inhibited HO-1 protein and failed to inhibit HO-2 protein. Incubation of endothelial cells with HO-1 antisense enhanced heme-dependent increase of ICAM-1. In contrast, addition of HO-2 antisense to endothelial cells failed to increase adhesion molecules. The role of glutathione, an important antioxidant, was examined on heme

  7. Gene Turnover in the Avian Globin Gene Families and Evolutionary Changes in Hemoglobin Isoform Expression

    PubMed Central

    Opazo, Juan C.; Hoffmann, Federico G.; Natarajan, Chandrasekhar; Witt, Christopher C.; Berenbrink, Michael; Storz, Jay F.

    2015-01-01

    The apparent stasis in the evolution of avian chromosomes suggests that birds may have experienced relatively low rates of gene gain and loss in multigene families. To investigate this possibility and to explore the phenotypic consequences of variation in gene copy number, we examined evolutionary changes in the families of genes that encode the α- and β-type subunits of hemoglobin (Hb), the tetrameric α2β2 protein responsible for blood-O2 transport. A comparative genomic analysis of 52 bird species revealed that the size and membership composition of the α- and β-globin gene families have remained remarkably constant during approximately 100 My of avian evolution. Most interspecific variation in gene content is attributable to multiple independent inactivations of the αD-globin gene, which encodes the α-chain subunit of a functionally distinct Hb isoform (HbD) that is expressed in both embryonic and definitive erythrocytes. Due to consistent differences in O2-binding properties between HbD and the major adult-expressed Hb isoform, HbA (which incorporates products of the αA-globin gene), recurrent losses of αD-globin contribute to among-species variation in blood-O2 affinity. Analysis of HbA/HbD expression levels in the red blood cells of 122 bird species revealed high variability among lineages and strong phylogenetic signal. In comparison with the homologous gene clusters in mammals, the low retention rate for lineage-specific gene duplicates in the avian globin gene clusters suggests that the developmental regulation of Hb synthesis in birds may be more highly conserved, with orthologous genes having similar stage-specific expression profiles and similar functional properties in disparate taxa. PMID:25502940

  8. Expression of thyroid hormone receptor isoforms down-regulated by thyroid hormone in human medulloblastoma cells.

    PubMed

    Monden, Tsuyoshi; Nakajima, Yasuyo; Hashida, Tetsu; Ishii, Sumiyasu; Tomaru, Takuya; Shibusawa, Nobuyuki; Hashimoto, Koshi; Satoh, Teturou; Yamada, Masanobu; Mori, Masatomo; Kasai, Kikuo

    2006-04-01

    The role of thyroid hormone (T3) in the regulation of growth and development of the central nervous system including the cerebellum has been well established. However, the effects of thyroid hormone on malignant tumors derived from the cerebellum remain poorly understood. Our analysis mainly focused on expression levels of TR isoforms and the effects of thyroid hormone in human medulloblastoma HTB-185 cells. Northern blot analysis revealed TRalpha2 mRNA but not TRalpha1, beta1 or beta2 mRNA in the cell. The TRalpha1 and TRbeta1 mRNAs were detected only by RT-PCR method and TRbeta2 was not expressed. Incubation of T3 for 24 h decreased TRalpha1, TRalpha2 and TRbeta1 mRNA. Addition of actinomycin D caused an acute increase in the basal TR mRNA levels and the rate of decrease of all kinds of TR isoform mRNA was accelerated in the T3-treated groups compared to controls, indicating that the stability of TR mRNA was affected by T3. Incubation with cycloheximide also blocked a decrease in TR mRNA levels in the T3-treated HTB-185 cells suggesting that down-regulation of TR mRNA required the synthesis of new protein. Our data provide novel evidence for the expression of TRs down-regulated by T3 in HTB-185 cells, suggesting that TR expression is post-transcriptionally regulated by T3 at the level of RNA stability.

  9. Differential regulation of constitutive androstane receptor expression by hepatocyte nuclear factor4alpha isoforms.

    PubMed

    Pascussi, Jean Marc; Robert, Agnes; Moreau, Amelie; Ramos, Jeanne; Bioulac-Sage, Paulette; Navarro, Francis; Blanc, Pierre; Assenat, Eric; Maurel, Patrick; Vilarem, Marie Jose

    2007-05-01

    Constitutive androstane receptor (CAR; NR1I3) controls the metabolism and elimination of endogenous and exogenous toxic compounds by up-regulating a battery of genes. In this work, we analyzed the expression of human CAR (hCAR) in normal liver during development and in hepatocellular carcinoma (HCC) and investigated the effect of hepatocyte nuclear factor 4alpha isoforms (HNF4alpha1 and HNF4alpha7) on the hCAR gene promoter. By performing functional analysis of hCAR 5'-deletions including mutants, chromatin immunoprecipitation in human hepatocytes, electromobility shift and cotransfection assays, we identified a functional and species-conserved HNF4alpha response element (DR1: ccAGGCCTtTGCCCTga) at nucleotide -144. Both HNF4alpha isoforms bind to this element with similar affinity. However, HNF4alpha1 strongly enhanced hCAR promoter activity whereas HNF4alpha7 was a poor activator and acted as a repressor of HNF4alpha1-mediated transactivation of the hCAR promoter. PGC1alpha stimulated both HNF4alpha1-mediated and HNF4alpha7-mediated hCAR transactivation to the same extent, whereas SRC1 exhibited a marked specificity for HNF4alpha1. Transduction of human hepatocytes by HNF4alpha7-expressing lentivirus confirmed this finding. In addition, we observed a positive correlation between CAR and HNF4alpha1 mRNA levels in human liver samples during development, and an inverse correlation between CAR and HNF4alpha7 mRNA levels in HCC. These observations suggest that HNF4alpha1 positively regulates hCAR expression in normal developing and adult livers, whereas HNF4alpha7 represses hCAR gene expression in HCC.

  10. Gene turnover in the avian globin gene families and evolutionary changes in hemoglobin isoform expression.

    PubMed

    Opazo, Juan C; Hoffmann, Federico G; Natarajan, Chandrasekhar; Witt, Christopher C; Berenbrink, Michael; Storz, Jay F

    2015-04-01

    The apparent stasis in the evolution of avian chromosomes suggests that birds may have experienced relatively low rates of gene gain and loss in multigene families. To investigate this possibility and to explore the phenotypic consequences of variation in gene copy number, we examined evolutionary changes in the families of genes that encode the α- and β-type subunits of hemoglobin (Hb), the tetrameric α2β2 protein responsible for blood-O2 transport. A comparative genomic analysis of 52 bird species revealed that the size and membership composition of the α- and β-globin gene families have remained remarkably constant during approximately 100 My of avian evolution. Most interspecific variation in gene content is attributable to multiple independent inactivations of the α(D)-globin gene, which encodes the α-chain subunit of a functionally distinct Hb isoform (HbD) that is expressed in both embryonic and definitive erythrocytes. Due to consistent differences in O2-binding properties between HbD and the major adult-expressed Hb isoform, HbA (which incorporates products of the α(A)-globin gene), recurrent losses of α(D)-globin contribute to among-species variation in blood-O2 affinity. Analysis of HbA/HbD expression levels in the red blood cells of 122 bird species revealed high variability among lineages and strong phylogenetic signal. In comparison with the homologous gene clusters in mammals, the low retention rate for lineage-specific gene duplicates in the avian globin gene clusters suggests that the developmental regulation of Hb synthesis in birds may be more highly conserved, with orthologous genes having similar stage-specific expression profiles and similar functional properties in disparate taxa.

  11. Cellular Expression, Trafficking, and Function of Two Isoforms of Human ULBP5/RAET1G

    PubMed Central

    Eagle, Robert A.; Flack, Gillian; Warford, Anthony; Martínez-Borra, Jesús; Jafferji, Insiya; Traherne, James A.; Ohashi, Maki; Boyle, Louise H.; Barrow, Alexander D.; Caillat-Zucman, Sophie; Young, Neil T.; Trowsdale, John

    2009-01-01

    Background The activating immunoreceptor NKG2D is expressed on Natural Killer (NK) cells and subsets of T cells. NKG2D contributes to anti-tumour and anti-viral immune responses in vitro and in vivo. The ligands for NKG2D in humans are diverse proteins of the MIC and ULBP/RAET families that are upregulated on the surface of virally infected cells and tumours. Two splicing variants of ULBP5/RAET1G have been cloned previously, but not extensively characterised. Methodology/Principal Findings We pursue a number of approaches to characterise the expression, trafficking, and function of the two isoforms of ULBP5/RAET1G. We show that both transcripts are frequently expressed in cell lines derived from epithelial cancers, and in primary breast cancers. The full-length transcript, RAET1G1, is predicted to encode a molecule with transmembrane and cytoplasmic domains that are unique amongst NKG2D ligands. Using specific anti-RAET1G1 antiserum to stain tissue microarrays we show that RAET1G1 expression is highly restricted in normal tissues. RAET1G1 was expressed at a low level in normal gastrointestinal epithelial cells in a similar pattern to MICA. Both RAET1G1 and MICA showed increased expression in the gut of patients with celiac disease. In contrast to healthy tissues the RAET1G1 antiserum stained a wide variety or different primary tumour sections. Both endogenously expressed and transfected RAET1G1 was mainly found inside the cell, with a minority of the protein reaching the cell surface. Conversely the truncated splicing variant of RAET1G2 was shown to encode a soluble molecule that could be secreted from cells. Secreted RAET1G2 was shown to downregulate NKG2D receptor expression on NK cells and hence may represent a novel tumour immune evasion strategy. Conclusions/Significance We demonstrate that the expression patterns of ULBP5RAET1G are very similar to the well-characterised NKG2D ligand, MICA. However the two isoforms of ULBP5/RAET1G have very different cellular

  12. Pyruvate kinase isoform expression alters nucleotide synthesis to impact cell proliferation

    PubMed Central

    Lunt, Sophia Y.; Muralidhar, Vinayak; Hosios, Aaron M.; Israelsen, William J.; Gui, Dan Y.; Newhouse, Lauren; Ogrodzinski, Martin; Hecht, Vivian; Xu, Kali; Acevedo, Paula N. Marín; Hollern, Daniel P.; Bellinger, Gary; Dayton, Talya L.; Christen, Stefan; Elia, Ilaria; Dinh, Anh T.; Stephanopoulos, Gregory; Manalis, Scott R.; Yaffe, Michael B.; Andrechek, Eran R.; Fendt, Sarah-Maria; Heiden, Matthew G. Vander

    2014-01-01

    SUMMARY Metabolic regulation influences cell proliferation. The influence of pyruvate kinase isoforms on tumor cells has been extensively studied, but whether PKM2 is required for normal cell proliferation is unknown. We examine how PKM2-deletion affects proliferation and metabolism in non-transformed, non-immortalized PKM2-expressing primary cells. We find that deletion of PKM2 in primary cells results in PKM1 expression and proliferation arrest. PKM1 expression, rather than PKM2 loss, is responsible for this effect, and proliferation arrest cannot be explained by cell differentiation, senescence, death, changes in gene expression, or prevention of cell growth. Instead, PKM1 expression impairs nucleotide production and the ability to synthesize DNA and progress through the cell cycle. Nucleotide biosynthesis is limiting, as proliferation arrest is characterized by severe thymidine depletion, and supplying exogenous thymine rescues both nucleotide levels and cell proliferation. Thus, PKM1 expression promotes a metabolic state that is unable to support DNA synthesis. PMID:25482511

  13. Characterization of the expression of the pro-metastatic Mena(INV) isoform during breast tumor progression.

    PubMed

    Oudin, Madeleine J; Hughes, Shannon K; Rohani, Nazanin; Moufarrej, Mira N; Jones, Joan G; Condeelis, John S; Lauffenburger, Douglas A; Gertler, Frank B

    2016-03-01

    Several functionally distinct isoforms of the actin regulatory Mena are produced by alternative splicing during tumor progression. Forced expression of the Mena(INV) isoform drives invasion, intravasation and metastasis. However, the abundance and distribution of endogenously expressed Mena(INV) within primary tumors during progression remain unknown, as most studies to date have only assessed relative mRNA levels from dissociated tumor samples. We have developed a Mena(INV) isoform-specific monoclonal antibody and used it to examine Mena(INV) expression patterns in mouse mammary and human breast tumors. Mena(INV) expression increases during tumor progression and to examine the relationship between Mena(INV) expression and markers for epithelial or mesenchymal status, stemness, stromal cell types and hypoxic regions. Further, while Mena(INV) robustly expressed in vascularized areas of the tumor, it is not confined to cells adjacent to blood vessels. Altogether, these data demonstrate the specificity and utility of the anti-Mena(INV)-isoform specific antibody, and provide the first description of endogenous Mena(INV) protein expression in mouse and human tumors.

  14. Differential expression of myosin heavy chain isoforms in the masticatory muscles of dystrophin-deficient mice.

    PubMed

    Spassov, Alexander; Gredes, Tomasz; Gedrange, Tomasz; Lucke, Silke; Morgenstern, Sven; Pavlovic, Dragan; Kunert-Keil, Christiane

    2011-12-01

    The dystrophin-deficient mouse (mdx) is a homologue animal model of Duchenne muscular dystrophy (DMD) and is characterized by slowly progressive muscle weakness accompanied by changes in myosin heavy chain (MyHC) composition. It is likely that the masticatory muscles undergo similar changes. The aim of this study was to examine the masticatory muscles (masseter, temporal, tongue, and soleus) of 100-day-old mdx and control mice (n = 8-10), and the fibre type distribution (by immunohistochemistry) as well as the expression of the corresponding MyHC messenger RNA (mRNA) (protein and mRNA expression, using Western blot or quantitative real-time polymerase chain reaction (RT-PCR)). Immunohistochemistry and western blot analysis revealed that the masticatory muscles in the control and mdx mice consisted mainly of type 2 fibres, whereas soleus muscle consisted of both type 1 and 2 fibres. In the masseter muscle, the mRNA in mdx mice was not different from that found in the controls. However, the mRNA content of the MyHC-2b isoform in mdx mice was lower in comparison with the controls in the temporal muscle [11.9 versus 36.9 per cent; P < 0.01; mean ± standard error of the mean (SEM), Student's unpaired t-test], as well as in the tongue muscle (65.7 versus 73.8 per cent; P < 0.05). Similarly, the content of MyHC-2x isoforms in mdx tongue muscle was lower than in the controls (25.9 versus 30.8 per cent; P < 0.05). The observed down-regulation of the MyHC-2x and MyHC-2b mRNA in the masticatory muscles of mdx mice may lead to changed fibre type composition. The different MyHC gene expression in mdx mice masticatory muscles may be seen as an adaptive mechanism to muscular dystrophy.

  15. Muscular tissues of the squid Doryteuthis pealeii express identical myosin heavy chain isoforms: an alternative mechanism for tuning contractile speed.

    PubMed

    Shaffer, Justin F; Kier, William M

    2012-01-15

    The speed of muscle contraction is largely controlled at the sarcomere level by the ATPase activity of the motor protein myosin. Differences in amino acid sequence in catalytically important regions of myosin yield different myosin isoforms with varying ATPase activities and resulting differences in cross-bridge cycling rates and interfilamentary sliding velocities. Modulation of whole-muscle performance by changes in myosin isoform ATPase activity is regarded as a universal mechanism to tune contractile properties, especially in vertebrate muscles. Invertebrates such as squid, however, may exhibit an alternative mechanism to tune contractile properties that is based on differences in muscle ultrastructure, including variable myofilament and sarcomere lengths. To determine definitively whether contractile properties of squid muscles are regulated via different myosin isoforms (i.e. different ATPase activities), the nucleotide and amino acid sequences of the myosin heavy chain from the squid Doryteuthis pealeii were determined from the mantle, arm, tentacle, fin and funnel retractor musculature. We identified three myosin heavy chain isoforms in squid muscular tissues, with differences arising at surface loop 1 and the carboxy terminus. All three isoforms were detected in all five tissues studied. These results suggest that the muscular tissues of D. pealeii express identical myosin isoforms, and it is likely that differences in muscle ultrastructure, not myosin ATPase activity, represent the most important mechanism for tuning contractile speeds.

  16. Isoform expression in the multiple soluble malate dehydrogenase of Hoplias malabaricus (Erythrinidae, Characiformes).

    PubMed

    Aquino-Silva, M R; Schwantes, M L; Schwantes, A R

    2003-02-01

    Kinetic properties and thermal stabilities of Hoplias malabaricus liver and skeletal muscle unfractionated malate dehydrogenase (MDH, EC 1.1.1.37) and its isolated isoforms were analyzed to further study the possible sMDH-A* locus duplication evolved from a recent tandem duplication. Both A (A1 and A2) and B isoforms had similar optima pH (7.5-8.0). While Hoplias A isoform could not be characterized as thermostable, B could as thermolabile. A isoforms differed from B isoform in having higher Km values for oxaloacetate. The possibly duplicated A2 isoform showed higher substrate affinity than the A1. Hoplias duplicated A isoforms may influence the direction of carbon flow between glycolisis and gluconeogenesis.

  17. Immunomodulatory pathways regulate expression of a spliced FKBP51 isoform in lymphocytes of melanoma patients.

    PubMed

    Romano, Simona; D'Angelillo, Anna; Staibano, Stefania; Simeone, Ester; D'Arrigo, Paolo; Ascierto, Paolo Antonio; Scalvenzi, Massimiliano; Mascolo, Massimo; Ilardi, Gennaro; Merolla, Francesco; Jovarauskaite, Egle; Romano, Maria Fiammetta

    2015-07-01

    FKBP51 (gene FKBP5) is an immunophilin capable of immunosuppression expressed in melanoma and lymphocytes. We found increased levels of a spliced FKBP5 variant in the PBMCs of 124 patients with melanoma. This variant encodes for an unknown isoform (FKBP51s). We hypothesized that FKBP51s resulted from tumour interaction with immune cells, through PDL-1/PD-1. To address this issue, we performed melanoma/PBMC cocultures. Furthermore, the immunohistochemistry of 76 melanoma specimens served to investigate whether FKBP51s stained tumour infiltrating lymphocytes. Our results showed that PBMCs expressed FKBP51s when cocultured with melanoma. Tumour PDL-1 knockdown or anti-PD-1 reduced FKBP51s expression in cocultured PBMCs. IHC showed a strong FKBP51s signal in tumour infiltrating lymphocytes, and lymphocytes of the invasion front of the tumour, along with melanoma PDL-1 expression. When overexpressed in melanoma, FKBP51s facilitated PDL-1 expression on the cell surface. In conclusion, our study shows that FKBP51s marks the PBMCs of patients with melanoma and is exploited by the tumour to immunomodulate through PDL-1.

  18. Differential expression of equine myosin heavy-chain mRNA and protein isoforms in a limb muscle.

    PubMed

    Eizema, Karin; van den Burg, Maarten; Kiri, Arpna; Dingboom, Elizabeth G; van Oudheusden, Hans; Goldspink, Geoffrey; Weijs, Wim A

    2003-09-01

    The horse is one of the few animals kept and bred for its athletic performance and is therefore an interesting model for human sports performance. The regulation of the development of equine locomotion in the first year of life, and the influence of early training on later performance, are largely unknown. The major structural protein in skeletal muscle, myosin heavy-chain (MyHC), is believed to be primarily transcriptionally controlled. To investigate the expression of the MyHC genes at the transcriptional level, we isolated cDNAs encoding the equine MyHC isoforms type 1 (slow), type 2a (fast oxidative), and type 2d/x (fast glycolytic). cDNAs encoding the 2b gene were not identified. The mRNA expression was compared to the protein expression on a fiber-to-fiber basis using in situ hybridization (non-radioactive) and immunohistochemistry. Marked differences were detected between the expression of MyHC transcripts and MyHC protein isoforms in adult equine gluteus medius muscle. Mismatches were primarily due to the presence of hybrid fibers expressing two fast (2ad) MyHC protein isoforms, but only one fast (mainly 2a) MyHC RNA isoform. This discrepancy was most likely not due to differential mRNA expression of myonuclei.

  19. Platelet amyloid precursor protein isoform expression in Alzheimer's disease: evidence for peripheral marker.

    PubMed

    Vignini, A; Sartini, D; Morganti, S; Nanetti, L; Luzzi, S; Provinciali, L; Mazzanti, L; Emanuelli, M

    2011-01-01

    Alzheimer's disease (AD) is a chronic neurodegenerative disorder characterized by a progressive cognitive and memory decline. Among peripheral markers of AD, great interest has been focused on the amyloid precursor protein (APP). In this regard, platelets represent an important peripheral source of APP since it has been demonstrated that the three major isoforms, that are constituted of 770, 751 and 695 aa residues, are inserted in the membrane of resting platelets. APP 751 and APP 770 contain a Kunitz-type serine protease inhibitor domain (APP KPI) and APP 695 lacks this domain. To address this issue, we first examined the platelet APP isoform mRNAs prospectively as biomarker for the diagnosis of AD by means of real-time quantitative PCR, and then evaluated the correlation between APP mRNA expression levels and cognitive impairment of enrolled subjects. Differential gene expression measurements in the AD patient group (n=18) revealed a significant up-regulation of APP TOT (1.52-fold), APP KPI (1.32-fold), APP 770 (1.33-fold) and APP 751 (1.26-fold) compared to controls (n=22). Moreover, a statistically significant positive correlation was found between APP mRNA levels (TOT, KPI, 770 and 751) and cognitive impairment. Since AD definitive diagnosis still relies on pathological evaluation at autopsy, the present results are consistent with the hypothesis that platelet APP could be considered a potential reliable peripheral marker for studying AD and could contribute to define a signature for the presence of AD pathology.

  20. Identification and expression of an atypical isoform of metallothionein in the African clawed frog Xenopus laevis.

    PubMed

    Scudiero, Rosaria; Tussellino, Margherita; Carotenuto, Rosa

    2015-05-01

    Exploiting the annotation of the western clawed frog Silurana tropicalis genome, we identified a new metallothionein (MT) gene, exhibiting all the features to be considered an active gene, but with an atypical coding region, showing only 17 cysteine residues instead of the canonical 20 cysteines of vertebrate metallothioneins and two anomalous cysteine triplets. However, the presence of a gene in the genome does not ensure its effective expression. By using conventional and Real-Time PCR analyses, we demonstrated that this atypical MT is constitutively expressed throughout the life cycle of the African clawed frog Xenopus laevis; moreover, this gene is highly expressed in the adult liver, the major site of MT expression and synthesis in vertebrates. To our knowledge, the X. laevis MT described in this paper is the first sequence of a vertebrate MT showing only 17 cysteine residues, arranged in two Cys-Cys-Cys motifs. Phylogenetic analyses also demonstrated that the atypical X. laevis MT merges in the anuran clade, but is the most derived sequence among tetrapods MTs. Finally, Tajima's Relative Rate Test suggested a different evolutionary rate between the canonical X. laevis MT and this novel isoform.

  1. Distinct cytochrome P450 aromatase isoforms in the common carp (Cyprinus carpio): sexual dimorphism and onset of ontogenic expression.

    PubMed

    Barney, Megan L; Patil, Jawahar G; Gunasekera, Rasanthi M; Carter, Chris G

    2008-05-01

    Cytochrome P450 aromatase (CYP19) is a key enzyme in the steroidogenic pathway that catalyses the conversion of testosterone to estrogen, and therefore is thought to influence gonadal sex differentiation. In an effort to understand the role of this enzyme in ovarian differentiation, we isolated cDNA encoding the two distinct isoforms, ovarian and brain (termed cyp19a and cyp19b, respectively) of adult common carp, Cyprinus carpio. The cloned cDNA for cyp19a had an open reading frame (ORF) of 518 amino acid residues, in contrast to cyp19b with an ORF of 511 amino acids. Sequence and phylogenetic analysis showed that these CYP19 isoforms were orthologous with previously described cyp19a and cyp19b from other teleosts. Quantitative real-time PCR indicated that both isoforms are expressed in adult ovary and brain, with predominant expression of cyp19a in the ovary and cyp19b in the brain. The major aromatase expressing tissue was found to be the brain, with greatest cyp19b expression in the anterior quarter (telencephalon) in both sexes. The gonad showed sexually dimorphic expression of both genes and dimorphic expression of cyp19a was observed in the cerebellum and the liver. Ontogenic expression showed that only the ovarian aromatase transcript is inherited maternally, with lower expression observed through early larval development under warmer rearing conditions. The differential and overlapping expression suggests these two aromatase genes have different roles in reproductive physiology.

  2. Upregulation of functional Kv11.1 isoform expression by inhibition of intronic polyadenylation with antisense morpholino oligonucleotides.

    PubMed

    Gong, Qiuming; Stump, Matthew R; Zhou, Zhengfeng

    2014-11-01

    The KCNH2 gene encodes the Kv11.1 potassium channel that conducts the rapidly activating delayed rectifier current in the heart. KCNH2 pre-mRNA undergoes alternative processing; intron 9 splicing leads to the formation of a functional, full-length Kv11.1a isoform, while polyadenylation within intron 9 generates a non-functional, C-terminally truncated Kv11.1a-USO isoform. The relative expression of Kv11.1 isoforms plays an important role in the regulation of Kv11.1 channel function and the pathogenesis of long QT syndrome. In this study, we identified cis-acting elements that are required for KCNH2 intron 9 poly(A) signal activity. Mutation of these elements decreased Kv11.1a-USO expression and increased the expression of Kv11.1a mRNA, protein and channel current. More importantly, blocking these elements by antisense morpholino oligonucleotides shifted the alternative processing of KCNH2 intron 9 from the polyadenylation to the splicing pathway, leading to the predominant production of Kv11.1a and a significant increase in Kv11.1 current. Our findings indicate that the expression of the Kv11.1a isoform can be upregulated by an antisense approach. Antisense inhibition of KCNH2 intronic polyadenylation represents a novel approach to increase Kv11.1 channel function.

  3. Glucose transporter isoform 1 expression enhances metastasis of malignant melanoma cells

    PubMed Central

    Koch, Andreas; Lang, Sven Arke; Wild, Peter Johannes; Gantner, Susanne; Mahli, Abdo; Spanier, Gerrit; Berneburg, Mark; Müller, Martina; Bosserhoff, Anja Katrin; Hellerbrand, Claus

    2015-01-01

    The glucose transporter isoform 1 (GLUT1; SLC2A1) is a key rate-limiting factor in the transport of glucose into cancer cells. Enhanced GLUT1 expression and accelerated glycolysis have been found to promote aggressive growth in a range of tumor entities. However, it was unknown whether GLUT1 directly impacts metastasis. Here, we aimed at analyzing the expression and function of GLUT1 in malignant melanoma. Immunohistochemical analysis of 78 primary human melanomas on a tissue micro array showed that GLUT1 expression significantly correlated with the mitotic activity and a poor survival. To determine the functional role of GLUT1 in melanoma, we stably suppressed GLUT1 in the murine melanoma cell line B16 with shRNA. GLUT1 suppressed melanoma cells revealed significantly reduced proliferation, apoptosis resistance, migratory activity and matrix metalloproteinase 2 (MMP2) expression. In a syngeneic murine model of hepatic metastasis, GLUT1-suppressed cells formed significantly less metastases and showed increased apoptosis compared to metastases formed by control cells. Treatment of four different human melanoma cell lines with a pharmacological GLUT1 inhibitor caused a dose-dependent reduction of proliferation, apoptosis resistance, migratory activity and MMP2 expression. Analysis of MAPK signal pathways showed that GLUT1 inhibition significantly decreased JNK activation, which regulates a wide range of targets in the metastatic cascade. In summary, our study provides functional evidence that enhanced GLUT1 expression in melanoma cells favors their metastatic behavior. These findings specify GLUT1 as an attractive therapeutic target and prognostic marker for this highly aggressive tumor. PMID:26293674

  4. Altered expression of pectoral myosin heavy chain isoforms corresponds to migration status in the white-crowned sparrow (Zonotrichia leucophrys gambelii)

    PubMed Central

    Welch, Kenneth C.; Ramenofsky, Marilyn

    2016-01-01

    Birds undergo numerous changes as they progress through life-history stages, yet relatively few studies have examined how birds adapt to both the dynamic energetic and mechanical demands associated with such transitions. Myosin heavy chain (MyHC) expression, often linked with muscle fibre type, is strongly correlated with a muscle's mechanical power-generating capability, thus we examined several morphological properties, including MyHC expression of the pectoralis, in a long-distance migrant, the white-crowned sparrow (Zonotrichia leucophrys gambelii) throughout the progression from winter, spring departure and arrival on breeding grounds. White-crowned sparrows demonstrated significant phenotypic flexibility throughout the seasonal transition, including changes in prealternate moult status, lipid fuelling, body condition and flight muscle morphology. Pectoral MyHC expression also varied significantly over the course of the study. Wintering birds expressed a single, newly classified adult fast 2 isoform. At spring departure, pectoral isoform expression included two MyHC isoforms: the adult fast 2 isoform along with a smaller proportion of a newly present adult fast 1 isoform. By spring arrival, both adult fast isoforms present at departure remained, yet expression had shifted to a greater relative proportion of the adult fast 1 isoform. Altering pectoral MyHC isoform expression in preparation for and during spring migration may represent an adaptation to modulate muscle mechanical output to support long-distance flight. PMID:28018664

  5. Immunohistochemical Expression of Heparanase Isoforms and Syndecan-1 Proteins in Colorectal Adenomas

    PubMed Central

    Waisberg, J.; Theodoro, T.R.; Matos, L.L.; Brasil, F.; Serrano, R.L.; Saba, G.T.; Pinhal, M.A.S.

    2016-01-01

    The proteoglycan syndecan-1 and the endoglucuronidases heparanase-1 and heparanase-2 are involved in molecular pathways that deregulate cell adhesion during carcinogenesis. Few studies have examined the expression of syndecan-1, heparanase-1 and mainly heparanase-2 proteins in non-neoplastic and neoplastic human colorectal adenoma tissues. The aim of this study was to analyze the correlation among the heparanase isoforms and the syndecan-1 proteins through immunohistochemical expression in the tissue of colorectal adenomas. Primary antihuman polyclonal anti-HPSE and anti-HPSE2 antibodies and primary anti-human monoclonal anti-SDC1 antibody were used in the immunohistochemical study. The expressions of heparanase-1 and heparanase-2 proteins were determined in tissue samples from 65 colorectal adenomas; the expression of syndecan-1 protein was obtained from 39 (60%) patients. The histological type of adenoma was tubular in 44 (67.7%) patients and tubular-villous in 21 (32.3%); there were no villous adenomas. The polyps were <1.0 cm in size in 54 (83.1%) patients and ≥1.0 cm in 11 (16.9%). The images were quantified by digital counter with a computer program for this purpose. The expression index represented the relationship between the intensity expression and the percentage of positively stained cells. The results showed that the average of heparanase-1, heparanase-2 and syndecan-1 expression index was 73.29 o.u./µm², 93.34 o.u./µm², and 55.29 o.u./µm², respectively. The correlation between the heparanase-1 and syndecan-1 expression index was positive (R=0.034) and significant (P=0.035). There was a negative (R= -0.384) and significant (P=0.016) correlation between the expression index of heparanase-1 and heparanase-2. A negative (R= -0.421) and significant (P=0.008) correlation between the expression index of heparanase-2 and syndecan-1 was found. We concluded that in colorectal adenomas, the heparanase-1 does not participate in syndecan-1 degradation; the

  6. [Isoforms of the human histamine H3 receptor: Generation, expression in the central nervous system and functional implications].

    PubMed

    García-Gálvez, Ana Maricela; Arias-Montaño, José Antonio

    2016-01-01

    Histamine plays a significant role as a neuromodulator in the human central nervous system. Histamine-releasing neurons are exclusively located in the tuberomammillary nucleus of the hypothalamus, project to all major areas of the brain, and participate in functions such as the regulation of sleep/wakefulness, locomotor activity, feeding and drinking, analgesia, learning, and memory. The functional effects of histamine are exerted through the activation of four G protein-coupled receptors (H1, H2, H3 and H4), and in the central nervous system the first three receptors are widely expressed. The H3 receptor (H3R) is found exclusively in neuronal cells, where it functions as auto- and hetero-receptor. One remarkable characteristic of the H3R is the existence of isoforms, generated by alternative splicing of the messenger RNA. For the human H3R, 20 isoforms have been reported; although a significant number lack those regions required for agonist binding or receptor signaling, at least five isoforms appear functional upon heterologous expression. In this work we review the evidence for the generation of human H3R isoforms, their expression, and the available information regarding the functionality of such receptors.

  7. Distinct Transcript Isoforms of the Atypical Chemokine Receptor 1 (ACKR1) / Duffy Antigen Receptor for Chemokines (DARC) Gene Are Expressed in Lymphoblasts and Altered Isoform Levels Are Associated with Genetic Ancestry and the Duffy-Null Allele

    PubMed Central

    Davis, Melissa B.; Walens, Andrea; Hire, Rupali; Mumin, Kauthar; Brown, Andrea M.; Ford, DeJuana; Howerth, Elizabeth W.; Monteil, Michele

    2015-01-01

    The Atypical ChemoKine Receptor 1 (ACKR1) gene, better known as Duffy Antigen Receptor for Chemokines (DARC or Duffy), is responsible for the Duffy Blood Group and plays a major role in regulating the circulating homeostatic levels of pro-inflammatory chemokines. Previous studies have shown that one common variant, the Duffy Null (Fy-) allele that is specific to African Ancestry groups, completely removes expression of the gene on erythrocytes; however, these individuals retain endothelial expression. Additional alleles are associated with a myriad of clinical outcomes related to immune responses and inflammation. In addition to allele variants, there are two distinct transcript isoforms of DARC which are expressed from separate promoters, and very little is known about the distinct transcriptional regulation or the distinct functionality of these protein isoforms. Our objective was to determine if the African specific Fy- allele alters the expression pattern of DARC isoforms and therefore could potentially result in a unique signature of the gene products, commonly referred to as antigens. Our work is the first to establish that there is expression of DARC on lymphoblasts. Our data indicates that people of African ancestry have distinct relative levels of DARC isoforms expressed in these cells. We conclude that the expression of both isoforms in combination with alternate alleles yields multiple Duffy antigens in ancestry groups, depending upon the haplotypes across the gene. Importantly, we hypothesize that DARC isoform expression patterns will translate into ancestry-specific inflammatory responses that are correlated with the axis of pro-inflammatory chemokine levels and distinct isoform-specific interactions with these chemokines. Ultimately, this work will increase knowledge of biological mechanisms underlying disparate clinical outcomes of inflammatory-related diseases among ethnic and geographic ancestry groups. PMID:26473357

  8. Recombinant proteinase 3 produced in different expression systems: recognition by anti-PR3 antibodies.

    PubMed

    van der Geld, Y M; Oost-Kort, W; Limburg, P C; Specks, U; Kallenberg, C G

    2000-10-20

    all anti-PR3 mAbs. In conclusion, rPR3 expressed in insect cells, HMC-1 and 293 cells is recognized by anti-PR3 antibodies, whereas conformational epitopes recognized by anti-PR3 mAbs and PR3-ANCA are not well preserved on rPR3 expressed in E. coli or P. pastoris.

  9. A novel isoform of the orphan nuclear receptor RORbeta is specifically expressed in pineal gland and retina.

    PubMed

    André, E; Gawlas, K; Becker-André, M

    1998-08-31

    RORbeta is a member of the nuclear hormone receptor superfamily whose ligand is unknown. Expression of RORbeta is confined to the central nervous system and its pattern suggests that this orphan nuclear receptor is implicated in the processing of sensory information and in circadian timing. In rats, RORbeta mRNA levels oscillate robustly in pineal gland and retina, displaying a 24h rhythm. Here we report the cloning of the cDNA of a novel isoform of RORbeta from rat pineal tissue. Expression of this isoform, called RORbeta2, is confined to pineal gland and retina and strongly increases at night. RORbeta2 shares common DNA- and putative ligand-binding domains with the canonical RORbeta (referred to as RORbeta1), but is characterized by a different amino-terminal domain. This structural difference renders RORbeta2 much more selectively binding to DNA than RORbeta1. Moreover, in contrast to RORbeta1, the novel isoform efficiently activates transcription also in non-neuronal cell lines. Thus, the two RORbeta isoforms are likely to regulate different sets of genes in different physiological contexts. 1998 Elsevier Science B.V.

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

    PubMed Central

    Marcel, Virginie; Cartet, Gaëlle; Lane, David P.; Lina, Bruno; Rosa-Calatrava, Manuel

    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 infection by using silencing and transient expression strategies in human lung epithelial cells. In addition, with the help of a panel of different influenza viruses from different subtypes, we also show that infection differentially regulates the expressions of p53β and Δ133p53α. Altogether, our results highlight the role of p53 isoforms in the viral cycle of influenza A viruses, with p53β and Δ133p53α acting as regulators of viral production in a p53-dependent manner. PMID:22647703

  11. Expression of a single ICAM-1 isoform on T cells is sufficient for development of experimental autoimmune encephalomyelitis.

    PubMed

    Bullard, Daniel C; Hu, Xianzhen; Crawford, David; McDonald, Kristin; Ramos, Theresa N; Barnum, Scott R

    2014-04-01

    Intercellular adhesion molecule-1 (ICAM-1) plays an important role in leukocyte trafficking, induction of cellular immune responses, and immunological synapse formation. As a member of the immunoglobulin superfamily of adhesion proteins, ICAM-1 is composed of repeating Ig-like domains, a transmembrane domain, and short cytoplasmic tail that participates in intracellular signaling events. At least seven ICAM-1 protein isoforms are generated by alternative splicing, however little is known regarding their immunobiology. We have previously shown using different lines of ICAM-1 mutant mice (Icam1(tm1Jcgr) and Icam1(tm1Bay) ) that expression of alternatively spliced ICAM-1 isoforms can significantly influence the disease course during the development of EAE. In this study, we show using a newly developed transgenic mouse (CD2-Icam1(D4del) /Icam1(null) ) that T-cell-specific expression of a single ICAM-1 isoform composed of Ig domains 1, 2, 3, and 5 can mediate the initiation and progression of EAE. Our results indicate that the ICAM-1 isoform lacking Ig domain 4 can drive pathogenesis in demyelinating disease and may be a novel therapeutic target for treating multiple sclerosis.

  12. Expression of a single ICAM-1 isoform on T cells is sufficient for development of experimental autoimmune encephalomyelitis

    PubMed Central

    Bullard, Daniel C.; Hu, Xianzhen; Crawford, David; McDonald, Kristen; Ramos, Theresa N.; Barnum, Scott R.

    2014-01-01

    Summary Intercellular adhesion molecule-1 (ICAM-1) plays an important role in leukocyte trafficking, induction of cellular immune responses, and immunological synapse formation. As a member of the immunoglobulin superfamily of adhesion proteins, ICAM-1 is composed of repeating Ig-like domains, a transmembrane domain, and short cytoplasmic tail that participates in intracellular signaling events. At least seven ICAM-1 protein isoforms are generated by alternative splicing, however little is known regarding their immunobiology. We have previously shown using different lines of ICAM-1 mutant mice (Icam1tm1Jcgr and Icam1tm1Bay) that expression of alternatively spliced ICAM-1 isoforms can significantly influence the disease course during the development of EAE. In this study, we show using a newly developed transgenic mouse (CD2-Icam1D4del/Icam1null) that T cell-specific expression of a single ICAM-1 isoform composed of Ig domains 1, 2, 3 and 5, can mediate the initiation and progression of EAE. Our results indicate that the ICAM-1 isoform lacking Ig domain 4 can drive pathogenesis in demyelinating disease and may be a novel therapeutic target for treating multiple sclerosis. PMID:24435747

  13. Estrogens Induce Expression of Membrane-Associated Estrogen Receptor α Isoforms in Lactotropes

    PubMed Central

    Zárate, Sandra; Jaita, Gabriela; Ferraris, Jimena; Eijo, Guadalupe; Magri, María L.; Pisera, Daniel; Seilicovich, Adriana

    2012-01-01

    Estrogens are key to anterior pituitary function, stimulating hormone release and controlling cell fate to achieve pituitary dynamic adaptation to changing physiological conditions. In addition to their classical mechanism of action through intracellular estrogen receptors (ERs), estrogens exert rapid actions via cell membrane-localized ERs (mERs). We previously showed that E2 exerts a rapid pro-apoptotic action in anterior pituitary cells, especially in lactotropes and somatotropes, through activation of mERs. In the present study, we examined the involvement of mERα in the rapid pro-apoptotic action of estradiol by TUNEL in primary cultures of anterior pituitary cells from ovariectomized rats using a cell-impermeable E2 conjugate (E2-BSA) and an ERα selective antagonist (MPP dihydrochloride). We studied mERα expression during the estrous cycle and its regulation by gonadal steroids in vivo by flow cytometry. We identified ERα variants in the plasma membrane of anterior pituitary cells during the estrous cycle and studied E2 regulation of these mERα variants in vitro by surface biotinylation and Western Blot. E2-BSA-induced apoptosis was abrogated by MPP in total anterior pituitary cells and lactotropes. In cycling rats, we detected a higher number of lactotropes and a lower number of somatotropes expressing mERα at proestrus than at diestrus. Acute E2 treatment increased the percentage of mERα-expressing lactotropes whereas it decreased the percentage of mERα-expressing somatotropes. We detected three mERα isoforms of 66, 39 and 22 kDa. Expression of mERα66 and mERα39 was higher at proestrus than at diestrus, and short-term E2 incubation increased expression of these two mERα variants. Our results indicate that the rapid apoptotic action exerted by E2 in lactotropes depends on mERα, probably full-length ERα and/or a 39 kDa ERα variant. Expression and activation of mERα variants in lactotropes could be one of the mechanisms through which E2

  14. Na+-dependent nucleoside transport in liver: two different isoforms from the same gene family are expressed in liver cells.

    PubMed Central

    Felipe, A; Valdes, R; Santo, B; Lloberas, J; Casado, J; Pastor-Anglada, M

    1998-01-01

    Hepatocytes show a Na+-dependent nucleoside transport activity that is kinetically heterogeneous and consistent with the expression of at least two independent concentrative Na+-coupled nucleoside transport systems (Mercader et al. Biochem. J. 317, 835-842, 1996). So far, only a single nucleoside carrier-related cDNA (SPNT) has been isolated from liver cells (Che et al. J. Biol. Chem. 270, 13596-13599, 1995). This cDNA presumably encodes a plasma membrane protein responsible for Na+-dependent purine nucleoside transport activity. Thus, the liver must express, at least, a second nucleoside transporter which should be pyrimidine-preferring. Homology cloning using RT-PCR revealed that a second isoform is indeed present in liver. This second isoform turned out to be identical to the 'epithelial-specific isoform' called cNT1, which shows in fact high specificity for pyrimidine nucleosides. Although cNT1 mRNA is present at lower amounts than SPNT mRNA, the amounts of cNT1 protein, when measured using isoform-specific polyclonal antibodies, were even higher than the SPNT protein levels. Moreover, partially purified basolateral plasma membrane vesicles from liver were enriched in the SPNT but not in the cNT1 protein, which suggests that the subcellular localization of these carrier proteins is different. SPNT and cNT1 protein amounts in crude membrane extracts from 6 h-regenerating rat livers are higher than in the preparations from sham-operated controls (3.5- and 2-fold, respectively). These results suggest that liver parenchymal cells express at least two different isoforms of concentrative nucleoside carriers, the cNT1 and SPNT proteins, which show differential regulation and subcellular localization. PMID:9480921

  15. The effects of phosphoramidon on the expression of human endothelin-converting enzyme-1 (ECE-1) isoforms.

    PubMed

    Isaka, Daiji; Emoto, Noriaki; Raharjo, Sunu Budhi; Yokoyama, Mitsuhiro; Matsuo, Masafumi

    2003-07-01

    Endothelin-1 (ET-1) is generated from big ET-1 by endothelin converting enzyme-1 (ECE-1). This process is inhibited by phosphoramidon through binding to the catalytic domain of ECE-1. There are four isoforms of human ECE-1 (ECE-1a, ECE-1b, ECE-1c and ECE-1d) which possess a conserved catalytic domain. Interestingly, a recent study has shown that in ECE-1b-transfected CHO cells phosphoramidon increases the expression and activity of ECE-1b. It is not known, however, whether phosphoramidon has similar effects on the expression of other ECE-1 isoforms. To address this point, we have established recombinant CHO cell lines that permanently express either human ECE-1a, ECE-1b or ECE-1c. Incubation of CHO/ECE-1a, -1b, and -1c with phosphoramidon (100 microM) for 16 hours markedly elevated the intracellular expression of ECE-1a and ECE-1b, but not ECE-1c protein, as indicated by Western blotting and immunocytochemistry. These increases appear to be due to inhibition of intracellular degradation of the protein because metabolic labeling followed by immunoprecipitation showed ECE-1a and ECE-1b proteins had prolonged half-lives in the phosphoramidon-treated cells. This is further supported by the finding that ECE-1 mRNA levels were unchanged following phosphoramidon treatment. Taken together, our results demonstrate that phosphoramidon differentially affects the expression of three human ECE-1 isoforms.

  16. Expression of eight glucocorticoid receptor isoforms in the human preterm placenta vary with fetal sex and birthweight

    PubMed Central

    Saif, Z.; Hodyl, N.A.; Stark, M.J.; Fuller, P.J.; Cole, T.; Lu, N.; Clifton, V.L.

    2016-01-01

    Introduction Administration of betamethasone to women at risk of preterm delivery is known to be associated with reduced fetal growth via alterations in placental function and possibly direct effects on the fetus. The placental glucocorticoid receptor (GR) is central to this response and recent evidence suggests there are numerous isoforms for GR in term placentae. In this study we have questioned whether GR isoform expression varies in preterm placentae in relation to betamethasone exposure, fetal sex and birthweight. Methods Preterm (24–36 completed weeks of gestation, n = 55) and term placentae (>37 completed weeks of gestation, n = 56) were collected at delivery. Placental GR expression was examined using Western Blot and analysed in relation to gestational age at delivery, fetal sex, birthweight and beta-methasone exposure. Data was analysed using non-parametric tests. Results Eight known isoforms of the GR were detected in the preterm placenta and include GRα (94 kDa), GRβ (91 kDa), GRα C (81 kDa) GR P (74 kDa) GR A (65 kDa), GRα D1–3 (50–55 kDa). Expression varied between preterm and term placentae with a greater expression of GRα C in preterm placentae relative to term placentae. The only sex differences in preterm placentae was that GRα D2 expression was higher in males than females. There were no alterations in preterm placental GR expression in association with betamethasone exposure. Discussion GRα C is the isoform involved in glucocorticoid induced apoptosis and suggests that its predominance in preterm placentae may contribute to the pathophysiology of preterm birth. PMID:25990415

  17. SLC44A2 single nucleotide polymorphisms, isoforms, and expression: Association with severity of Meniere's disease?

    PubMed

    Nair, Thankam S; Kommareddi, Pavan K; Galano, Maria M; Miller, Danielle M; Kakaraparthi, Bala Naveen; Telian, Steven A; Arts, H Alex; El-Kashlan, Hussam; Kilijanczyk, Alyse; Lassig, Amy Anne D; Graham, Martin P; Fisher, Susan G; Stoll, Stefan W; Nair, Rajan P; Elder, James T; Carey, Thomas E

    2016-12-01

    SLC44A2 was discovered as the target of an antibody that causes hearing loss. Knockout mice develop age related hearing loss, loss of sensory cells and spiral ganglion neurons. SLC44A2 has polymorphic sites implicated in human disease. Transfusion related acute lung injury (TRALI) is linked to rs2288904 and genome wide association studies link rs2288904 and rs9797861 to venous thromboembolism (VTE), coronary artery disease and stroke. Here we report linkage disequilibrium of rs2288904 with rs3087969 and the association of these SLC44A2 SNPs with Meniere's disease severity. Tissue-specific isoform expression differences suggest that the N-terminal domain is linked to different functions in different cell types. Heterozygosity at rs2288904 CGA/CAA and rs3087969 GAT/GAC showed a trend for association with intractable Meniere's disease compared to less severe disease and to controls. The association of SLC44A2 SNPs with VTE suggests that thrombi affecting cochlear vessels could be a factor in Meniere's disease.

  18. Molecular cloning, expression and immunological characterisation of Lol p 5C, a novel allergen isoform of rye grass pollen demonstrating high IgE reactivity.

    PubMed

    Suphioglu, C; Mawdsley, D; Schäppi, G; Gruehn, S; de Leon, M; Rolland, J M; O'Hehir, R E

    1999-12-03

    A novel isoform of a major rye grass pollen allergen Lol p 5 was isolated from a cDNA expression library. The new isoform, Lol p 5C, shares 95% amino acid sequence identity with Lol p 5A. Both isoforms demonstrated shared antigenic activity but different allergenic activities. Recombinant Lol p 5C demonstrated 100% IgE reactivity in 22 rye grass pollen sensitive patients. In comparison, recombinant Lol p 5A showed IgE reactivity in less than 64% of the patients. Therefore, Lol p 5C represents a novel and highly IgE-reactive isoform allergen of rye grass pollen.

  19. Differential Expression of Metallothionein Isoforms in Terrestrial Snail Embryos Reflects Early Life Stage Adaptation to Metal Stress

    PubMed Central

    Baurand, Pierre-Emmanuel; Pedrini-Martha, Veronika; de Vaufleury, Annette; Niederwanger, Michael; Capelli, Nicolas; Scheifler, Renaud; Dallinger, Reinhard

    2015-01-01

    The aim of this study was to analyze the expression of three metallothionein (MT) isoform genes (CdMT, CuMT and Cd/CuMT), already known from adults, in the Early Life Stage (ELS) of Cantareus aspersus. This was accomplished by detection of the MT isoform-specific transcription adopting Polymerase Chain Reaction (PCR) amplification and quantitative Real Time (qRT)-PCR of the three MT genes. Freshly laid eggs were kept for 24 hours under control conditions or exposed to three cadmium (Cd) solutions of increasing concentration (5, 10, and 15 mg Cd/L). The transcription of the three MT isoform genes was detected via PCR in 1, 6 and 12-day-old control or Cd-exposed embryos. Moreover, the transcription of this isoform genes during development was followed by qRT-PCR in 6 and 12-day-old embryos. Our results showed that the CdMT and Cd/CuMT genes, but not the CuMT gene, are expressed in embryos at the first day of development. The transcription of the 3 MT genes in control embryos increased with development time, suggesting that the capacities of metal regulation and detoxification may have gradually increased throughout embryogenesis. However in control embryos, the most highly expressed MT gene was that of the Cd/CuMT isoform, whose transcription levels greatly exceeded those of the other two MT genes. This contrasts with the minor significance of this gene in adult snails and suggests that in embryos, this isoform may play a comparatively more important role in metal physiology compared to adult individuals. This function in adult snails appears not to be related to Cd detoxification. Instead, snail embryos responded to Cd exposure by over-expression of the CdMT gene in a concentration-dependent manner, whereas the expression of the Cd/CuMT gene remained unaffected. Moreover, our study demonstrates the ability of snail embryos to respond very early to Cd exposure by up-regulation of the CdMT gene. PMID:25706953

  20. Diversified expression of NG2/CSPG4 isoforms in glioblastoma and human foetal brain identifies pericyte subsets.

    PubMed

    Girolamo, Francesco; Dallatomasina, Alice; Rizzi, Marco; Errede, Mariella; Wälchli, Thomas; Mucignat, Maria Teresa; Frei, Karl; Roncali, Luisa; Perris, Roberto; Virgintino, Daniela

    2013-01-01

    NG2/CSPG4 is a complex surface-associated proteoglycan (PG) recognized to be a widely expressed membrane component of glioblastoma (WHO grade IV) cells and angiogenic pericytes. To determine the precise expression pattern of NG2/CSPG4 on glioblastoma cells and pericytes, we generated a panel of >60 mouse monoclonal antibodies (mAbs) directed against the ectodomain of human NG2/CSPG4, partially characterized the mAbs, and performed a high-resolution distributional mapping of the PG in human foetal, adult and glioblastoma-affected brains. The reactivity pattern initially observed on reference tumour cell lines indicated that the mAbs recognized 48 immunologically distinct NG2/CSPG4 isoforms, and a total of 14 mAbs was found to identify NG2/CSPG4 isoforms in foetal and neoplastic cerebral sections. These were consistently absent in the adult brain, but exhibited a complementary expression pattern in angiogenic vessels of both tumour and foetal tissues. Considering the extreme pleomorphism of tumour areas, and with the aim of subsequently analysing the distributional pattern of the NG2/CSPG4 isoforms on similar histological vessel typologies, a preliminary study was carried out with endothelial cell and pericyte markers, and with selected vascular basement membrane (VBM) components. On both tumour areas characterized by 'glomeruloid' and 'garland vessels', which showed a remarkably similar cellular and molecular organization, and on developing brain vessels, spatially separated, phenotypically diversified pericyte subsets with a polarized expression of key surface components, including NG2/CSPG4, were disclosed. Interestingly, the majority of the immunolocalized NG2/CSPG4 isoforms present in glioblastoma tissue were present in foetal brain, except for one isoform that seemed to be exclusive of tumour cells, being absent in foetal brain. The results highlight an unprecedented, complex pattern of NG2/CSPG4 isoform expression in foetal and neoplastic CNS, discriminating

  1. Human Renal Normal, Tumoral, and Cancer Stem Cells Express Membrane-Bound Interleukin-15 Isoforms Displaying Different Functions1

    PubMed Central

    Azzi, Sandy; Gallerne, Cindy; Romei, Cristina; Le Coz, Vincent; Gangemi, Rosaria; Khawam, Krystel; Devocelle, Aurore; Gu, Yanhong; Bruno, Stefania; Ferrini, Silvano; Chouaib, Salem; Eid, Pierre; Azzarone, Bruno; Giron-Michel, Julien

    2015-01-01

    Intrarenal interleukin-15 (IL-15) participates to renal pathophysiology, but the role of its different membrane-bound isoforms remains to be elucidated. In this study, we reassess the biology of membrane-bound IL-15 (mb-IL-15) isoforms by comparing primary cultures of human renal proximal tubular epithelial cells (RPTEC) to peritumoral (ptumTEC), tumoral (RCC), and cancer stem cells (CSC/CD105+). RPTEC express a 14 to 16 kDa mb-IL-15, whose existence has been assumed but never formally demonstrated and likely represents the isoform anchored at the cell membrane through the IL-15 receptor α (IL-15Rα) chain, because it is sensitive to acidic treatment and is not competent to deliver a reverse signal. By contrast, ptumTEC, RCC, and CSC express a novel N-hyperglycosylated, short-lived transmembrane mb-IL-15 (tmb-IL-15) isoform around 27 kDa, resistant to acidic shock, delivering a reverse signal in response to its soluble receptor (sIL-15Rα). This reverse signal triggers the down-regulation of the tumor suppressor gene E-cadherin in ptumTEC and RCC but not in CSC/CD105+, where it promotes survival. Indeed, through the AKT pathway, tmb-IL-15 protects CSC/CD105+ from non-programmed cell death induced by serum starvation. Finally, both mb-IL-15 and tmb-IL-15 are sensitive to metalloproteases, and the cleaved tmb-IL-15 (25 kDa) displays a powerful anti-apoptotic effect on human hematopoietic cells. Overall, our data indicate that both mb-IL-15 and tmb-IL-15 isoforms play a complex role in renal pathophysiology downregulating E-cadherin and favoring cell survival. Moreover, “apparently normal” ptumTEC cells, sharing different properties with RCC, could contribute to organize an enlarged peritumoral “preneoplastic” environment committed to favor tumor progression. PMID:26152359

  2. Molecular characterization and expression profiles of four transformer-2 isoforms in the Chinese mitten crab Eriocheir sinensis

    NASA Astrophysics Data System (ADS)

    Luo, Danli; Liu, Yuan; Hui, Min; Song, Chengwen; Liu, Hourong; Cui, Zhaoxia

    2016-09-01

    The transformer-2 (tra-2) gene plays a key role in the regulatory hierarchy of sexual differentiation in somatic tissues and in the germline of Drosophila melanogaster. In this study, sequences and expression profiles of tra-2 in the Chinese mitten crab Eriocheir sinensis were characterized. Four tra-2 isoforms, designated as Estra-2a, Estra-2b, Estra-2c and Estra-2d, were isolated. They all contained an RNA-recognition motif (RRM) and a linker region, which shared high similarity with other reported tra-2s. Sequence analysis revealed that Estra-2a, Estra-2b and Estra-2c are encoded by the same genomic locus and are generated by alternative splicing of the pre-mRNA. Compared with the other three isoforms, Estra-2d lacks the RS2 domain. Quantitative real-time PCR showed that all four isoforms were highly expressed in the fertilized egg, and in the 2-4 cell and blastula stages compared with larval stages (P≤ 0.01), suggesting their maternal origin in early embryonic developmental stages. Notably, Estra-2a was highly expressed in male somatic tissues, while Estra-2c was significantly highly expressed in the ovary. These results suggest that Estra-2c is involved in sexual differentiation of the Chinese mitten crab. Our findings provide basic information for further functional studies of the tra-2 gene/protein in this species.

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

    PubMed Central

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

    2016-01-01

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

  4. Co-Expression of SERCA Isoforms, Phospholamban and Sarcolipin in Human Skeletal Muscle Fibers

    PubMed Central

    Fajardo, Val A.; Bombardier, Eric; Vigna, Chris; Devji, Tahira; Bloemberg, Darin; Gamu, Daniel; Gramolini, Anthony O.; Quadrilatero, Joe; Tupling, A. Russell

    2013-01-01

    Sarcolipin (SLN) and phospholamban (PLN) inhibit the activity of sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) by reducing their apparent affinity for Ca2+. A ternary complex between SLN, PLN, and SERCAs results in super-inhibition of SERCA activity. Analysis of skeletal muscle homogenate has limited our current understanding of whether SLN and PLN regulate SERCA1a, SERCA2a, or both in skeletal muscle and whether SLN and PLN are co-expressed in skeletal muscle fibers. Biopsies from human vastus lateralis were analyzed through single fiber Western blotting and immunohisto/fluorescence staining to circumvent this limitation. With a newly generated SLN antibody, we report for the first time that SLN protein is present in human skeletal muscle. Addition of the SLN antibody (50 µg) to vastus lateralis homogenates increased the apparent Ca2+ affinity of SERCA (KCa, pCa units) (-Ab, 5.85 ± 0.02 vs. +Ab, 5.95 ± 0.02) and maximal SERCA activity (μmol/g protein/min) (-Ab, 122 ± 6.4 vs. +Ab, 159 ± 11) demonstrating a functional interaction between SLN and SERCAs in human vastus lateralis. Specifically, our results suggest that although SLN and PLN may preferentially regulate SERCA1a, and SERCA2a, respectively, physiologically they both may regulate either SERCA isoform. Furthermore, we show that SLN and PLN co-immunoprecipitate in human vastus lateralis homogenate and are simultaneously expressed in 81% of the fibers analyzed with Western blotting which implies that super-inhibition of SERCA may exist in human skeletal muscle. Finally, we demonstrate unequivocally that mouse soleus contains PLN protein suggesting that super-inhibition of SERCA may also be important physiologically in rodent skeletal muscle. PMID:24358354

  5. Molecular characterization of two metallothionein isoforms in avian species: evolutionary history, tissue distribution profile, and expression associated with metal accumulation.

    PubMed

    Nam, Dong-Ha; Kim, Eun-Young; Iwata, Hisato; Tanabe, Shinsuke

    2007-04-01

    To characterize avian MTs, MT cDNAs were cloned from liver of cormorant (Phalacrocorax carbo) and mallard (Anas platyrhynchos). Expression profiles of MT isoforms and relationships between metal accumulation and MT mRNA expression in tissues were also investigated. We succeeded in cDNA cloning of MT1/2 from cormorant and MT1 in mallard. DNA sequence of chicken MT1 was obtained from chicken (Gallus gallus) genomic database. Considering previous reports on avian MTs, birds possess at least two distinct MT isoforms. Comparison of genomic synteny among vertebrates and phylogenetic analysis of MT amino acid sequences revealed that avian MT1/2 are evolutionarily close to mammalian MT3. Messenger RNAs of both MT isoforms were detected in all the tissues/organs in cormorant and mallard. Liver was the primary organ for cormorant MT1/2, and mallard MT2, whereas MT1 was dominant in mallard heart. Interspecies comparison of tissue distribution of MT mRNA expression between cormorant and mallard indicated that MT2 profile was similar, but MT1 was not. Significant positive correlations of mRNA expression levels between MT1 and MT2 were observed in the liver and kidney of cormorants, whereas no correlation was found in mallards. Expression levels of cormorant MT1/2 showed significant positive correlations with hepatic Cu and Zn concentrations, suggesting that both MT isoforms were induced by Cu and Zn in livers. Cormorant MT2 expression level exhibited a significant positive correlation with hepatic Ag, and a negative correlation with Rb, indicating that Ag and Rb concentrations depend on the expression of MT2 by Cu and Zn. In mallard, MT1 had no correlation with any metal concentration, and MT2 expression was positively correlated only with Cu, even though hepatic Cu and Zn concentrations in mallard were much higher than in cormorant. This may indicate that cormorant is a more susceptible species than mallard in terms of MT induction. These findings suggest tissue-, species

  6. Two isoforms of Saccharomyces cerevisiae glutaredoxin 2 are expressed in vivo and localize to different subcellular compartments.

    PubMed Central

    Pedrajas, José R; Porras, Pablo; Martínez-Galisteo, Emilia; Padilla, C Alicia; Miranda-Vizuete, Antonio; Bárcena, J Antonio

    2002-01-01

    Glutaredoxin (Grx)2 from Saccharomyces cerevisiae is a member of the two-cysteine (dithiol) subfamily of Grxs involved in the defence against oxidative stress in yeast. Recombinant yeast Grx2p, expressed in Escherichia coli, behaves as a 'classical' Grx that efficiently catalyses the reduction of hydroxyethyl disulphide by GSH. Grx2p also catalyses the reduction of GSSG by dihydrolipoamide with even higher efficiency. Western blot analysis of S. cerevisiae crude extracts identifies two isoforms of Grx2p of 15.9 and 11.9 kDa respectively. The levels of these two isoforms reach a peak during the exponential phase of growth in normal yeast extract/peptone/dextrose ('YPD') medium, with the long form predominating over the short one. From immunochemical analysis of subcellular fractions, it is shown that both isoforms are present in mitochondria, but only the short one is detected in the cytosolic fraction. On the other hand, only the long form is prominent in microsomes. Mitochondrial isoforms should represent the processed and unprocessed products of an open reading frame (YDR513W), with a putative start codon 99 bp upstream of the GRX2 start codon described thus far. These results indicate that GRX2 contains two in-frame start codons, and that translation from the first AUG results in a product that is targeted to mitochondria. The cytosolic form would result either by initiation from the second AUG, or by differential processing of one single translation product. PMID:11958675

  7. Differential expression of espin isoforms during epithelial morphogenesis, stereociliogenesis and postnatal maturation in the developing inner ear.

    PubMed

    Sekerková, Gabriella; Zheng, Lili; Mugnaini, Enrico; Bartles, James R

    2006-03-01

    The espins are a family of multifunctional actin cytoskeletal proteins. They are present in hair cell stereocilia and are the target of mutations that cause deafness and vestibular dysfunction. Here, we demonstrate that the different espin isoforms are expressed in complex spatiotemporal patterns during inner ear development. Espin 3 isoforms were prevalent in the epithelium of the otic pit, otocyst and membranous labyrinth as they underwent morphogenesis. This espin was down-regulated ahead of hair cell differentiation and during neuroblast delamination. Espin also accumulated in the epithelium of branchial clefts and pharyngeal pouches and during branching morphogenesis in other embryonic epithelial tissues, suggesting general roles for espins in epithelial morphogenesis. Espin reappeared later in inner ear development in differentiating hair cells. Its levels and compartmentalization to stereocilia increased during the formation and maturation of stereociliary bundles. Late in embryonic development, espin was also present in a tail-like process that emanated from the hair cell base. Increases in the levels of espin 1 and espin 4 isoforms correlated with stereocilium elongation and maturation in the vestibular system and cochlea, respectively. Our results suggest that the different espin isoforms play specific roles in actin cytoskeletal regulation during epithelial morphogenesis and hair cell differentiation.

  8. Temporal expression and mitochondrial localization of a Foxp2 isoform lacking the forkhead domain in developing Purkinje cells.

    PubMed

    Tanabe, Yuko; Fujiwara, Yuji; Matsuzaki, Ayumi; Fujita, Eriko; Kasahara, Tadashi; Yuasa, Shigeki; Momoi, Takashi

    2012-07-01

    FOXP2, a forkhead box-containing transcription factor, forms homo- or hetero-dimers with FOXP family members and localizes to the nucleus, while FOXP2(R553H), which contains a mutation related to speech/language disorders, features reduced DNA binding activity and both cytoplasmic and nuclear localization. In addition to being a loss-of-function mutation, it is possible that FOXP2(R553H) also may act as a gain-of-function mutation to inhibit the functions of FOXP2 isoforms including FOXP2Ex10+ lacking forkhead domain. Foxp2(R552H) knock-in mouse pups exhibit impaired ultrasonic vocalization and poor dendritic development in Purkinje cells. However, expressions of Foxp2 isoforms in the developing Purkinje are unclear. The appearance of 'apical cytoplasmic swelling' (mitochondria-rich regions that are the source of budding processes) correlates with dendritic development of Purkinje cells. In the present study, we focused on Foxp2 isoforms localizing to the apical cytoplasmic swelling and identified two isoforms lacking forkhead domain: Foxp2Ex12+ and Foxp2Ex15. They partly localized to the membrane fraction that includes mitochondria. Foxp2Ex12+ mainly localized to the apical cytoplasmic swelling in early developing Purkinje cells at the stellate stage (P2-P4). Mitochondrial localization of Foxp2Ex12+ in Purkinje cells was confirmed by immune-electron microscopic analysis. Foxp2Ex12+ may play a role in dendritic development in Purkinje cells.

  9. Bile Salt Export Pump is Dysregulated with Altered Farnesoid X Receptor Isoform Expression in Patients with Hepatocellular Carcinoma

    PubMed Central

    Chen, Yuan; Song, Xiulong; Valanejad, Leila; Vasilenko, Alexander; More, Vijay; Qiu, Xi; Chen, Weikang; Lai, Yurong; Slitt, Angela; Stoner, Matthew; Yan, Bingfang; Deng, Ruitang

    2012-01-01

    As a canalicular bile acid effluxer, bile salt export pump (BSEP) plays a vital role in maintaining bile acid homeostasis. BSEP deficiency leads to severe cholestasis and hepatocellular carcinoma (HCC) in young children. Regardless of the etiology, chronic inflammation is the common pathological process for HCC development. Clinical studies showed that bile acid homeostasis is disrupted in HCC patients with elevated serum bile acid level as a proposed marker for HCC. However, the underlying mechanisms remain largely unknown. In this study, we found that BSEP expression was severely diminished in HCC tissues and markedly reduced in adjacent non-tumor tissues. In contrast to mouse, human BSEP was regulated by farnesoid x receptor (FXR) in an isoform-dependent manner. FXRα2 exhibited a much more potent activity than FXRα1 in transactivating human BSEP in vitro and in vivo. The decreased BSEP expression in HCC was associated with altered relative expression of FXRα1 and FXRα2. The FXRα1/FXRα2 ratios were significantly increased with undetectable FXRα2 expression in one third of the HCC tumor samples. Similar correlation between BSEP and FXR isoform expression was confirmed in hepatoma Huh 7 and HepG2 cells. Further studies showed that intrahepatic proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were significantly elevated in HCC tissues. Treatment of Huh 7 cells with IL-6 and TNF-α resulted in a marked increase in the FXRα1/FXRα2 ratio concurrent with a significant decrease in BSEP expression. In conclusion, BSEP expression was severely diminished in HCC patients associated with alteration of FXR isoform expression induced by inflammation, and the restoration of BSEP expression through suppressing inflammation in the liver may re-establish the bile acid homeostasis. PMID:23213087

  10. Myosin isoform expression in the prehensile tails of didelphid marsupials: functional differences between arboreal and terrestrial opossums.

    PubMed

    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 derbianus (arboreal) and Monodelphis domestica (terrestrial), using a combination of gel electrophoresis and immunohistochemistry analyses. C. derbianus expresses three MHC isoforms (1, 2A, 2X) that are distributed (mean percentage) as 8.2% MHC-1, 2.6% 1/2A, and 89.2% 2A/X hybrid fibers. M. domestica also expresses MHC-1, 2A, and 2X, in addition to the 2B isoform, distributed as 17.0% MHC-1, 1.3% 1/2A, 9.0% 2A, 75.2% 2A/X, and 0.3% 2X/B hybrid fibers. The distribution of similar isoform fiber types differed significantly between species (P < 0.001). Although not statistically significant, C. derbianus was observed to have larger cross-sectional area (CSA) for each corresponding fiber type along with a greater amount of extra-cellular matrix. An overall faster fiber type composition (and larger fibers) in the tail of an arboreal specialist supports our hypothesis, and correlates with higher muscle force required for tail hanging and arboreal maneuvering on terminal substrates. Conversely, a broader distribution of highly oxidative fibers in the caudal musculature is well suited for

  11. DNMT3B7, a truncated DNMT3B isoform expressed in human tumors, disrupts embryonic development and accelerates lymphomagenesis

    PubMed Central

    Shah, Mrinal Y.; Vasanthakumar, Aparna; Barnes, Natalie Y.; Figueroa, Maria E.; Kamp, Anna; Hendrick, Christopher; Ostler, Kelly R.; Davis, Elizabeth M.; Lin, Shang; Anastasi, John; Le Beau, Michelle M.; Moskowitz, Ivan; Melnick, Ari; Pytel, Peter; Godley, Lucy A.

    2010-01-01

    Epigenetic changes are among the most common alterations observed in cancer cells, yet the mechanism by which cancer cells acquire and maintain abnormal DNA methylation patterns is not understood. Cancer cells have an altered distribution of DNA methylation and express aberrant DNA methyltransferase 3B transcripts, which encode truncated proteins, some of which lack the C-terminal catalytic domain. To test if a truncated DNMT3B isoform disrupts DNA methylation in vivo, we constructed two lines of transgenic mice expressing DNMT3B7, a truncated DNMT3B isoform commonly found in cancer cells. DNMT3B7 transgenic mice exhibit altered embryonic development, including lymphopenia, craniofacial abnormalities, and cardiac defects, similar to Dnmt3b-deficient animals, but rarely develop cancer. However, when DNMT3B7 transgenic are bred with Eμ-Myc transgenic mice, which model aggressive B cell lymphoma, DNMT3B7 expression increases the frequency of mediastinal lymphomas in Eμ-Myc animals. Eμ-Myc/DNMT3B7 mediastinal lymphomas have more chromosomal rearrangements, increased global DNA methylation levels, and more locus-specific perturbations in DNA methylation patterns compared to Eμ-Myc lymphomas. These data represent the first in vivo modeling of cancer-associated DNA methylation changes and suggest that truncated DNMT3B isoforms contribute to the re-distribution of DNA methylation characterizing virtually every human tumor. PMID:20587527

  12. Expression of progesterone receptor membrane component 1, serpine mRNA binding protein 1 and nuclear progesterone receptor isoforms A and B in the bovine myometrium during the estrous cycle and early pregnancy.

    PubMed

    Slonina, Dominika; Kowalik, Magdalena K; Kotwica, Jan

    2012-01-01

    The aim of this study was to investigate the (1) expression of progesterone membrane component 1 (PGRMC1), serpine mRNA binding protein 1 (SERBP1) and progesterone receptor (PR) mRNA and (2) protein expression levels of PGRMC1, SERBP1 and PR isoforms A and B in the bovine myometrium during the estrous cycle and early pregnancy. Uteri from cows on days 1-5, 6-10, 11-16 and 17-21 of the estrous cycle and weeks 3-5, 6-8 and 9-12 of pregnancy were used (n=5-6 per period). There were no changes (P>0.05) in PGRMC1 mRNA expression during the estrous cycle, while expression of SERBP1 and PR mRNA was the lowest (P<0.05) on days 11-16 relative to other days of the cycle. The highest mRNA expression of PGRMC1, SERBP1 and PR was found during pregnancy. There were no changes (P>0.05) in SERBP1 protein expression in cycling and pregnant cows, while the highest (P<0.05) PGRMC1 protein expression was found during weeks 3-5 of pregnancy. Similar protein expression profiles for PRA and PRB were found, and protein levels were highest on days 1-5 of the estrous cycle. From day 6 of the cycle, PRA and PRB protein expression decreased and were maintained at this lower level during pregnancy. In conclusion, our study assessed mRNA and protein expression levels of PGRMC1, SERBP1 and PR in the bovine myometrium during the estrous cycle and the first trimester of pregnancy. It is possible that progesterone (P4) affects myometrial function in a genomic and nongenomic manner.

  13. Novel exons in the tbx5 gene locus generate protein isoforms with distinct expression domains and function.

    PubMed

    Yamak, Abir; Georges, Romain O; Sheikh-Hassani, Massomeh; Morin, Martin; Komati, Hiba; Nemer, Mona

    2015-03-13

    TBX5 is the gene mutated in Holt-Oram syndrome, an autosomal dominant disorder with complex heart and limb deformities. Its protein product is a member of the T-box family of transcription factors and an evolutionarily conserved dosage-sensitive regulator of heart and limb development. Understanding TBX5 regulation is therefore of paramount importance. Here we uncover the existence of novel exons and provide evidence that TBX5 activity may be extensively regulated through alternative splicing to produce protein isoforms with differing N- and C-terminal domains. These isoforms are also present in human heart, indicative of an evolutionarily conserved regulatory mechanism. The newly identified isoforms have different transcriptional properties and can antagonize TBX5a target gene activation. Droplet Digital PCR as well as immunohistochemistry with isoform-specific antibodies reveal differential as well as overlapping expression domains. In particular, we find that the predominant isoform in skeletal myoblasts is Tbx5c, and we show that it is dramatically up-regulated in differentiating myotubes and is essential for myotube formation. Mechanistically, TBX5c antagonizes TBX5a activation of pro-proliferative signals such as IGF-1, FGF-10, and BMP4. The results provide new insight into Tbx5 regulation and function that will further our understanding of its role in health and disease. The finding of new exons in the Tbx5 locus may also be relevant to mutational screening especially in the 30% of Holt-Oram syndrome patients with no mutations in the known TBX5a exons.

  14. Expression of a calpastatin transgene slows muscle wasting and obviates changes in myosin isoform expression during murine muscle disuse

    NASA Technical Reports Server (NTRS)

    Tidball, James G.; Spencer, Melissa J.

    2002-01-01

    Muscle wasting is a prominent feature of several systemic diseases, neurological damage and muscle disuse. The contribution of calpain proteases to muscle wasting in any instance of muscle injury or disease has remained unknown because of the inability to specifically perturb calpain activity in vivo. We have generated a transgenic mouse with muscle-specific overexpression of calpastatin, which is the endogenous inhibitor of calpains, and induced muscle atrophy by unloading hindlimb musculature for 10 days. Expression of the transgene resulted in increases in calpastatin concentration in muscle by 30- to 50-fold, and eliminated all calpain activity that was detectable on zymograms. Muscle fibres in ambulatory, transgenic mice were smaller in diameter, but more numerous, so that muscle mass did not differ between transgenic and non-transgenic mice. This is consistent with the role of the calpain-calpastatin system in muscle cell fusion that has been observed in vitro. Overexpression of calpastatin reduced muscle atrophy by 30 % during the 10 day unloading period. In addition, calpastatin overexpression completely prevented the shift in myofibrillar myosin content from slow to fast isoforms, which normally occurs in muscle unloading. These findings indicate that therapeutics directed toward regulating the calpain-calpastatin system may be beneficial in preventing muscle mass loss in muscle injury and disease.

  15. PGC-1 isoforms and their target genes are expressed differently in human skeletal muscle following resistance and endurance exercise

    PubMed Central

    Silvennoinen, Mika; Ahtiainen, Juha P; Hulmi, Juha J; Pekkala, Satu; Taipale, Ritva S; Nindl, Bradley C; Laine, Tanja; Häkkinen, Keijo; Selänne, Harri; Kyröläinen, Heikki; Kainulainen, Heikki

    2015-01-01

    The primary aim of the present study was to investigate the acute gene expression responses of PGC-1 isoforms and PGC-1α target genes related to mitochondrial biogenesis (cytochrome C), angiogenesis (VEGF-A), and muscle hypertrophy (myostatin), after a resistance or endurance exercise bout. In addition, the study aimed to elucidate whether the expression changes of studied transcripts were linked to phosphorylation of AMPK and MAPK p38. Nineteen physically active men were divided into resistance exercise (RE, n = 11) and endurance exercise (EE, n = 8) groups. RE group performed leg press exercise (10 × 10 RM, 50 min) and EE walked on a treadmill (∼80% HRmax, 50 min). Muscle biopsies were obtained from the vastus lateralis muscle before, 30 min, and 180 min after exercise. EE and RE significantly increased the gene expression of alternative promoter originated PGC-1α exon 1b- and 1bxs’-derived isoforms, whereas the proximal promoter originated exon 1a-derived transcripts were less inducible and were upregulated only after EE. Truncated PGC-1α transcripts were upregulated both after EE and RE. Neither RE nor EE affected the expression of PGC-1β. EE upregulated the expression of cytochrome C and VEGF-A, whereas RE upregulated VEGF-A and downregulated myostatin. Both EE and RE increased the levels of p-AMPK and p-MAPK p38, but these changes were not linked to the gene expression responses of PGC-1 isoforms. The present study comprehensively assayed PGC-1 transcripts in human skeletal muscle and showed exercise mode-specific responses thus improving the understanding of early signaling events in exercise-induced muscle adaptations. PMID:26438733

  16. Expression of the WT1 gene -KTS domain isoforms suppresses the invasive ability of human lung squamous cell carcinoma cells.

    PubMed

    Moriya, Shogo; Takiguchi, Masaki; Seki, Naohiko

    2008-02-01

    Although the WT1 gene was originally isolated as a tumor suppressor gene from Wilms' tumor, oncogenic roles for WT1 have been reported in several tumors. Here, we present new findings of high levels of WT1 expression associated with the suppression of lymph node metastasis in patients with human lung squamous cell carcinoma (SCC). We investigated the effect of down-regulated WT1 gene expression on the invasive phenotype of the SCC cell line RERF-LC-AI. Invasive ability was enhanced in WT1-specific siRNA-transfected cells, and a WT1 target gene p21(Waf1/Cip1) was isolated by comprehensive gene expression analysis. As several isoforms are produced from the WT1 gene, we isolated eight major WT1 isoforms from a cDNA library and cloned each variant into an expression vector. Luciferase reporter assays revealed that p21(Waf1/Cip1) expression was enhanced only by the WT1 cDNA variants that included a three-amino acid deletion (-KTS). Our results suggested that the -KTS-containing variants of WT1 are directly involved in the regulation of p21(Waf1/Cip1) expression and the subsequent suppression of lymph node metastasis in human lung squamous cell carcinoma.

  17. Transforming growth factor-{beta}1 regulates fibronectin isoform expression and splicing factor SRp40 expression during ATDC5 chondrogenic maturation

    SciTech Connect

    Han Fei; Gilbert, James R.; Harrison, Gerald; Adams, Christopher S.; Freeman, Theresa; Tao Zhuliang; Zaka, Raihana; Liang Hongyan; Williams, Charlene; Tuan, Rocky S.; Norton, Pamela A.; Hickok, Noreen J. . E-mail: Noreen.Hickok@jefferson.edu

    2007-05-01

    Fibronectin (FN) isoform expression is altered during chondrocyte commitment and maturation, with cartilage favoring expression of FN isoforms that includes the type II repeat extra domain B (EDB) but excludes extra domain A (EDA). We and others have hypothesized that the regulated splicing of FN mRNAs is necessary for the progression of chondrogenesis. To test this, we treated the pre-chondrogenic cell line ATDC5 with transforming growth factor-{beta}1, which has been shown to modulate expression of the EDA and EDB exons, as well as the late markers of chondrocyte maturation; it also slightly accelerates the early acquisition of a sulfated proteoglycan matrix without affecting cell proliferation. When chondrocytes are treated with TGF-{beta}1, the EDA exon is preferentially excluded at all times whereas the EDB exon is relatively depleted at early times. This regulated alternative splicing of FN correlates with the regulation of alternative splicing of SRp40, a splicing factor facilitating inclusion of the EDA exon. To determine if overexpression of the SRp40 isoforms altered FN and FN EDA organization, cDNAs encoding these isoforms were overexpressed in ATDC5 cells. Overexpression of the long-form of SRp40 yielded an FN organization similar to TGF-{beta}1 treatment; whereas overexpression of the short form of SRp40 (which facilitates EDA inclusion) increased formation of long-thick FN fibrils. Therefore, we conclude that the effects of TGF-{beta}1 on FN splicing during chondrogenesis may be largely dependent on its effect on SRp40 isoform expression.

  18. Opposing functions of TFII-I spliced isoforms in growth factor-induced gene expression.

    PubMed

    Hakre, Shweta; Tussie-Luna, María Isabel; Ashworth, Todd; Novina, Carl D; Settleman, Jeffrey; Sharp, Phillip A; Roy, Ananda L

    2006-10-20

    Multifunctional transcription factor TFII-I has two spliced isoforms (Delta and beta) in murine fibroblasts. Here we show that these isoforms have distinct subcellular localization and mutually exclusive transcription functions in the context of growth factor signaling. In the absence of signaling, TFII-Ibeta is nuclear and recruited to the c-fos promoter in vivo. But upon growth factor stimulation, the promoter recruitment is abolished and it is exported out of the nucleus. Moreover, isoform-specific silencing of TFII-Ibeta results in transcriptional activation of the c-fos gene. In contrast, TFII-IDelta is largely cytoplasmic in the resting state but translocates to the nucleus upon growth factor signaling, undergoes signal-induced recruitment to the same site on the c-fos promoter, and activates the gene. Importantly, activated TFII-IDelta interacts with Erk1/2 (MAPK) kinase in the cell cytoplasm and imports the Erk1/2 to the nucleus, thereby transducing growth factor signaling. Our results identify a unique growth factor signaling pathway controlled by opposing activities of two TFII-I spliced isoforms.

  19. Developmental Febrile Seizures Modulate Hippocampal Gene Expression of Hyperpolarization-Activated Channels in an Isoform- and Cell-Specific Manner

    PubMed Central

    Brewster, Amy; Bender, Roland A.; Chen, Yuncai; Dube, Celine; Eghbal-Ahmadi, Mariam; Baram, Tallie Z.

    2012-01-01

    Febrile seizures, in addition to being the most common seizure type of the developing human, may contribute to the generation of subsequent limbic epilepsy. Our previous work has demonstrated that prolonged experimental febrile seizures in the immature rat model increased hippocampal excitability long term, enhancing susceptibility to future seizures. The mechanisms for these profound proepileptogenic changes did not require cell death and were associated with long-term slowed kinetics of the hyperpolarization-activated depolarizing current (IH). Here we show that these seizures modulate the expression of genes encoding this current, the hyperpolarization-activated, cyclic nucleotide-gated channels (HCNs): In CA1 neurons expressing multiple HCN isoforms, the seizures induced a coordinated reduction of HCN1 mRNA and enhancement of HCN2 expression, thus altering the neuronal HCN phenotype. The seizure-induced augmentation of HCN2 expression involved CA3 in addition to CA1, whereas for HCN4, mRNA expression was not changed by the seizures in either hippocampal region. This isoform- and region-specific transcriptional regulation of the HCNs required neuronal activity rather than hyperthermia alone, correlated with seizure duration, and favored the formation of slow-kinetics HCN2-encoded channels. In summary, these data demonstrate a novel, activity-dependent transcriptional regulation of HCN molecules by developmental seizures. These changes result in long-lasting alteration of the HCN phenotype of specific hippocampal neuronal populations, with profound consequences on the excitability of the hippocampal network. PMID:12040066

  20. Developmental febrile seizures modulate hippocampal gene expression of hyperpolarization-activated channels in an isoform- and cell-specific manner.

    PubMed

    Brewster, Amy; Bender, Roland A; Chen, Yuncai; Dube, Celine; Eghbal-Ahmadi, Mariam; Baram, Tallie Z

    2002-06-01

    Febrile seizures, in addition to being the most common seizure type of the developing human, may contribute to the generation of subsequent limbic epilepsy. Our previous work has demonstrated that prolonged experimental febrile seizures in the immature rat model increased hippocampal excitability long term, enhancing susceptibility to future seizures. The mechanisms for these profound proepileptogenic changes did not require cell death and were associated with long-term slowed kinetics of the hyperpolarization-activated depolarizing current (I(H)). Here we show that these seizures modulate the expression of genes encoding this current, the hyperpolarization-activated, cyclic nucleotide-gated channels (HCNs): In CA1 neurons expressing multiple HCN isoforms, the seizures induced a coordinated reduction of HCN1 mRNA and enhancement of HCN2 expression, thus altering the neuronal HCN phenotype. The seizure-induced augmentation of HCN2 expression involved CA3 in addition to CA1, whereas for HCN4, mRNA expression was not changed by the seizures in either hippocampal region. This isoform- and region-specific transcriptional regulation of the HCNs required neuronal activity rather than hyperthermia alone, correlated with seizure duration, and favored the formation of slow-kinetics HCN2-encoded channels. In summary, these data demonstrate a novel, activity-dependent transcriptional regulation of HCN molecules by developmental seizures. These changes result in long-lasting alteration of the HCN phenotype of specific hippocampal neuronal populations, with profound consequences on the excitability of the hippocampal network.

  1. Molecular cloning and expression analysis of PDK family genes in Xenopus laevis reveal oocyte-specific PDK isoform.

    PubMed

    Terazawa, Yumiko; Tokmakov, Alexander A; Shirouzu, Mikako; Yokoyama, Shigeyuki

    2005-12-30

    Pyruvate dehydrogenase kinase (PDK) inactivates the multienzyme mitochondrial pyruvate dehydrogenase complex by the phosphorylation of three seryl residues in the pyruvate dehydrogenase moiety, and thus plays an important role in the control of glucose homeostasis. Genetically and biochemically distinct PDK family isozymes have been identified in mammalian species. In the present study, we demonstrate that the complete family of expressed PDK family genes in the tissues of the African clawed frog, Xenopus laevis, consists of four members, which are divided into two evolutionary groups. Xenopus PDKs (xPDKs) share an overall homology of about 70% to the human isoforms of PDK. The abundance of mRNAs for the four xPDK isoforms was analyzed by the real-time reverse transcriptase PCR technique in the various tissues of Xenopus laevis, including heart, lung, spleen, liver, kidney, skin, testis, oocytes, and eggs. Our data suggest that one of the xPDK isozymes can be referred to as an oocyte-specific xPDK. Functional differences between the xPDK isoforms are discussed, based on their different tissue-specific distributions and phylogenetic similarities to human PDKs.

  2. Expression of metallothoinein isoform 3 is restricted at the post-transcriptional level in human bladder epithelial cells.

    PubMed

    Garrett, Scott H; Park, Seongmi; Sens, Mary Ann; Somji, Seema; Singh, Rajendra K; Namburi, Venugopal B R K; Sens, Donald A

    2005-09-01

    This study was designed to define the effect that overexpression of MT-3 would have on a cell culture model of bladder urothelium. Stable and inducible transfection was used to achieve overexpression of the MT-3 gene in the UROtsa cell line. When the UROtsa cells were stably transfected with the MT-3 coding sequence, there was highly elevated expression of MT-3 mRNA, but no MT-3 protein. An inducible vector showed that low basal levels of MT-3 mRNA and protein could be produced, but that induction only increased MT-3 mRNA and not protein. The clones expressing low basal levels of MT-3 protein also had reduced growth rates compared to control cells. Site directed mutagenesis was used to produce an MT-3 coding sequence where the prolines in positions 7 and 9 were converted to threonines. When this altered MT-3 was stably transfected into the UROtsa cells, the cells were able to accumulate the mutated form of the MT-3 protein. These studies show that MT-3 protein expression is inhibited by post-transcriptional control in the urothelial cell. Modifying the MT-3 protein to resemble the MT-1 isoform removes this component of post-transcriptional control and allows accumulation of the mutated MT-3 protein. The altered sequence involved in post-transcriptional control of MT-3 protein expression is the same sequence implicated in the neuronal growth inhibitory activity associated specifically with the MT-3 isoform of the MT gene family.

  3. Silencing and heterologous expression of ppo-2 indicate a specific function of a single polyphenol oxidase isoform in resistance of dandelion (Taraxacum officinale) against Pseudomonas syringae pv. tomato.

    PubMed

    Richter, Carolin; Dirks, Mareike E; Gronover, Christian Schulze; Prüfer, Dirk; Moerschbacher, Bruno M

    2012-02-01

    Dandelion (Taraxacum officinale) possesses an unusually high degree of disease resistance. As this plant exhibits high polyphenol oxidase (PPO) activity and PPO have been implicated in resistance against pests and pathogens, we analyzed the potential involvement of five PPO isoenzymes in the resistance of dandelion against Botrytis cinerea and Pseudomonas syringae pv. tomato. Only one PPO (ppo-2) was induced during infection, and ppo-2 promoter and β-glucuronidase marker gene fusions revealed strong induction of the gene surrounding lesions induced by B. cinerea. Specific RNAi silencing reduced ppo-2 expression only, and concomitantly increased plant susceptibility to P. syringae pv. tomato. At 4 days postinoculation, P. syringae pv. tomato populations were strongly increased in the ppo-2 RNAi lines compared with wild-type plants. When the dandelion ppo-2 gene was expressed in Arabidopsis thaliana, a plant having no PPO gene, active protein was formed and protein extracts of the transgenic plants exhibited substrate-dependent antimicrobial activity against P. syringae pv. tomato. These results clearly indicate a strong contribution of a specific, single PPO isoform to disease resistance. Therefore, we propose that specific PPO isoenzymes be included in a new family of pathogenesis-related (PR) proteins.

  4. Evolutionarily conserved sequences of striated muscle myosin heavy chain isoforms. Epitope mapping by cDNA expression.

    PubMed

    Miller, J B; Teal, S B; Stockdale, F E

    1989-08-05

    A cDNA expression strategy was used to localize amino acid sequences which were specific for fast, as opposed to slow, isoforms of the chicken skeletal muscle myosin heavy chain (MHC) and which were conserved in vertebrate evolution. Five monoclonal antibodies (mAbs), termed F18, F27, F30, F47, and F59, were prepared that reacted with all of the known chicken fast MHC isoforms but did not react with any of the known chicken slow nor with smooth muscle MHC isoforms. The epitopes recognized by mAbs F18, F30, F47, and F59 were on the globular head fragment of the MHC, whereas the epitope recognized by mAb F27 was on the helical tail or rod fragment. Reactivity of all five mAbs also was confined to fast MHCs in the rat, with the exception of mAb F59, which also reacted with the beta-cardiac MHC, the single slow MHC isoform common to both the rat heart and skeletal muscle. None of the five epitopes was expressed on amphioxus, nematode, or Dictyostelium MHC. The F27 and F59 epitopes were found on shark, electric ray, goldfish, newt, frog, turtle, chicken, quail, rabbit, and rat MHCs. The epitopes recognized by these mAbs were conserved, therefore, to varying degrees through vertebrate evolution and differed in sequence from homologous regions of a number of invertebrate MHCs and myosin-like proteins. The sequence of those epitopes on the head were mapped using a two-part cDNA expression strategy. First, Bal31 exonuclease digestion was used to rapidly generate fragments of a chicken embryonic fast MHC cDNA that were progressively deleted from the 3' end. These cDNA fragments were expressed as beta-galactosidase/MHC fusion proteins using the pUR290 vector; the fusion proteins were tested by immunoblotting for reactivity with the mAbs; and the approximate locations of the epitopes were determined from the sizes of the cDNA fragments that encoded a particular epitope. The epitopes were then precisely mapped by expression of overlapping cDNA fragments of known sequence that

  5. PR genes of apple: identification and expression in response to elicitors and inoculation with Erwinia amylovora

    PubMed Central

    Bonasera, Jean M; Kim, Jihyun F; Beer, Steven V

    2006-01-01

    Background In the past decade, much work has been done to dissect the molecular basis of the defence signalling pathway in plants known as Systemic Acquired Resistance (SAR). Most of the work has been carried out in model species such as Arabidopsis, with little attention paid to woody plants. However within the range of species examined, components of the pathway seem to be highly conserved. In this study, we attempted to identify downstream components of the SAR pathway in apple to serve as markers for its activation. Results We identified three pathogenesis related (PR) genes from apple, PR-2, PR-5 and PR-8, which are induced in response to inoculation with the apple pathogen, Erwinia amylovora, but they are not induced in young apple shoots by treatment with known elicitors of SAR in herbaceous plants. We also identified three PR-1-like genes from apple, PR-1a, PR-1b and PR-1c, based solely on sequence similarity to known PR-1 genes of model (intensively researched) herbaceous plants. The PR-1-like genes were not induced in response to inoculation with E. amylovora or by treatment with elicitors; however, each showed a distinct pattern of expression. Conclusion Four PR genes from apple were partially characterized. PR-1a, PR-2, PR-5 and PR-8 from apple are not markers for SAR in young apple shoots. Two additional PR-1-like genes were identified through in-silico analysis of apple ESTs deposited in GenBank. PR-1a, PR-1b and PR-1c are not involved in defence response or SAR in young apple shoots; this conclusion differs from that reported previously for young apple seedlings. PMID:17029637

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

    PubMed

    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

    2015-10-15

    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 a very low false positive rate. The results show that IDP-fusion will be useful for unraveling the complexity of multiple fusion splices and fusion isoforms within tumorigenesis-relevant fusion genes.

  7. POU5F1 isoforms show different expression patterns in human embryonic stem cells and preimplantation embryos.

    PubMed

    Cauffman, Greet; Liebaers, Inge; Van Steirteghem, André; Van de Velde, Hilde

    2006-12-01

    The contribution of the POU domain, class 5, transcription factor-1 (POU5F1) in maintaining totipotency in human embryonic stem cells (hESCs) has been repeatedly proven. In humans, two isoforms are encoded: POU5F1_iA and POU5F1_iB. So far, no discrimination has been made between the isoforms in POU5F1 studies, and it is unknown which isoform contributes to the undifferentiated phenotype. Using immunocytochemistry, expression of POU5F1_iA and POU5F1_iB was examined in hESCs and all stages of human preimplantation development to look for differences in expression, biological activity, and relation to totipotency. POU5F1_iA and POU5F1_iB displayed different temporal and spatial expression patterns. During human preimplantation development, a significant POU5F1_iA expression was seen in all nuclei of compacted embryos and blastocysts and a clear POU5F1_iB expression was detected from the four-cell stage onwards in the cytoplasm of all cells. The cytoplasmic localization might imply no or other biological functions beyond transcription activation for POU5F1_iB. The stemness properties of POU5F1 can be assigned to POU5F1_iA because hESCs expressed POU5F1_iA but not POU5F1_iB. However, POU5F1_iA is not the appropriate marker to identify totipotent cells, because POU5F1_iA was also expressed in the nontotipotent trophectoderm and was not expressed in zygotes and early cleavage stage embryos, which are assumed to be totipotent. The expression pattern of POU5F1_iA may suggest that POU5F1_iA alone cannot sustain totipotency and that coexpression with other stemness factors might be the key to totipotency.

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

    PubMed

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

    2015-08-01

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

  9. Divergent prion strain evolution driven by PrPC expression level in transgenic mice

    PubMed Central

    Le Dur, Annick; Laï, Thanh Lan; Stinnakre, Marie-George; Laisné, Aude; Chenais, Nathalie; Rakotobe, Sabine; Passet, Bruno; Reine, Fabienne; Soulier, Solange; Herzog, Laetitia; Tilly, Gaëlle; Rézaei, Human; Béringue, Vincent; Vilotte, Jean-Luc; Laude, Hubert

    2017-01-01

    Prions induce a fatal neurodegenerative disease in infected host brain based on the refolding and aggregation of the host-encoded prion protein PrPC into PrPSc. Structurally distinct PrPSc conformers can give rise to multiple prion strains. Constrained interactions between PrPC and different PrPSc strains can in turn lead to certain PrPSc (sub)populations being selected for cross-species transmission, or even produce mutation-like events. By contrast, prion strains are generally conserved when transmitted within the same species, or to transgenic mice expressing homologous PrPC. Here, we compare the strain properties of a representative sheep scrapie isolate transmitted to a panel of transgenic mouse lines expressing varying levels of homologous PrPC. While breeding true in mice expressing PrPC at near physiological levels, scrapie prions evolve consistently towards different strain components in mice beyond a certain threshold of PrPC overexpression. Our results support the view that PrPC gene dosage can influence prion evolution on homotypic transmission. PMID:28112164

  10. Dynamic expression of DNMT3a and DNMT3b isoforms during male germ cell development in the mouse.

    PubMed

    La Salle, Sophie; Trasler, Jacquetta M

    2006-08-01

    In the male germ line, sequence-specific methylation patterns are initially acquired prenatally in diploid gonocytes and are further consolidated after birth during spermatogenesis. It is still unclear how DNA methyltransferases are involved in establishing and/or maintaining these patterns in germ cells, or how their activity is regulated. We compared the temporal expression patterns of the postulated de novo DNA methyltransferases DNMT3a and DNMT3b in murine male germ cells. Mitotic, meiotic and post-meiotic male germ cells were isolated, and expression of various transcript variants and isoforms of Dnmt3a and Dnmt3b was examined using Quantitative RT-PCR and Western blotting. We found that proliferating and differentiating male germ cells were marked by distinctive expression profiles. Dnmt3a2 and Dnmt3b transcripts were at their highest levels in type A spermatogonia, decreased dramatically in type B spermatogonia and preleptotene spermatocytes and rose again in leptotene/zygotene spermatocytes, while Dnmt3a expression was mostly constant, except in type B spermatogonia where it increased. In all cases, expression declined as pachynema progressed. At the protein level, DNMT3a was the predominant isoform in type B spermatogonia, while DNMT3a2, DNMT3b2, and DNMT3b3 were expressed throughout most of spermatogenesis, except in pachytene spermatocytes. We also detected DNMT3a2 and DNMT3b2 in round spermatids. Taken together, these data highlight the tightly regulated expression of these genes during spermatogenesis and provide evidence that DNMTs may be contributing differentially to the establishment and/or maintenance of methylation patterns in male germ cells.

  11. Complex modulation of the expression of PKC isoforms in the rat brain during chronic type 1 diabetes mellitus.

    PubMed

    Vetri, Francesco; Chavez, Rafael; Xu, Hao-Liang; Paisansathan, Chanannait; Pelligrino, Dale A

    2013-01-15

    We previously demonstrated that chronic hyperglycemia has a detrimental influence on neurovascular coupling in the brain-an effect linked to an alteration in the protein kinase C (PKC)-mediated phosphorylation pattern. Moreover, the activity of PKC was increased, in diabetic rat brain, in a tissue fraction composed primarily of the superficial glia limitans and pial vessels, but trended toward a decrease in cerebral cortical gray matter. However, that study did not examine the expression patterns of PKC isoforms in the rat brain. Thus, in a rat model of streptozotocin (STZ)-induced chronic type 1 diabetes mellitus (T1DM), and in non-diabetic (ND) controls, two hypotheses were addressed. First, chronic T1DM is accompanied by changes in the expression of PKC-α, βII, γ, δ, and ε Second, those changes differ when comparing cerebral cortex and glio-pial tissue. In addition, we analyzed the expression of a form of PKC-γ, phosphorylated on threonine 514 (pT514-PKC-γ), as well as the receptor for activated C kinase 1 (RACK1). The expression pattern of different PKC isoforms was altered in a complex and tissue-specific manner during chronic hyperglycemia. Notably, in the gray matter, PKC-α expression significantly decreased, while pT514-PKC-γ expression increased. However, PKC-βII, -γ, -δ, -ε, and RACK1 expressions did not change. Conversely, in glio-pial tissue, PKC-α and RACK1 were upregulated, whereas PKC-γ, pT514-PKC-γ, and PKC-ε were downregulated. PKC-βII, and PKC-δ, were unchanged. These findings suggest that the PKC activity increase previously seen in the glio-pial tissue of diabetic rats may be due to the selective upregulation of PKC-α, and ultimately lead to the impairment of neurovascular coupling.

  12. Expression and regulation of pyruvate dehydrogenase kinase isoforms in the developing rat heart and in adulthood: role of thyroid hormone status and lipid supply.

    PubMed Central

    Sugden, M C; Langdown, M L; Harris, R A; Holness, M J

    2000-01-01

    Activation of the pyruvate dehydrogenase (PDH) complex (PDHC) promotes glucose disposal, whereas inactivation conserves glucose. The PDH kinases (PDHKs) regulate glucose oxidation through inhibitory phosphorylation of PDHC. The adult rat heart contains three PDHK isoforms PDHK1, PDHK2 and PDHK4. Using Western-blot analysis, with specific antibodies raised against individual recombinant PDHK1, PDHK2 and PDHK4, the present study investigated PDHK isoform expression in the developing rat heart and adulthood. We identified clear differences in the patterns of protein expression of each of these PDHK isoforms during the first 3 weeks of post-natal development, with most marked up-regulation of isoforms PDHK1 and PDHK4. Distinctions between the three cardiac PDHK isoforms were also demonstrated with respect to post-neonatal maturational up-regulation; with greatest up-regulation of PDHK1 and least up-regulation of PDHK4 from the post-neonatal period until maturity. The study also examined the role of thyroid hormone status and lipid supply on PDHK isoform expression. We observed marked selective increases in the amount of PDHK4 protein present relative to total cardiac protein in both hyperthyroidism and high-fat feeding. Overall, our data identify PDHK isoform PDHK1 as being of more potential regulatory importance for glucose oxidation in the adult compared with the neonatal heart, and cardiac PDHK4 as a PDHK isoform whose expression is specifically responsive to changes in lipid supply, suggesting that its up-regulation during early post-natal life may be the perinatal switch to use fatty acids as the energy source. We also identify regulation of pyruvate sensitivity of cardiac PDHK as a physiological variable, a change in which requires factors in addition to a change in lipid supply. PMID:11104680

  13. Alternative 5' exons and differential splicing regulate expression of protein 4.1R isoforms with distinct n-termini

    SciTech Connect

    Parra, Marilyn K.; Gee, Sherry L.; Koury, Mark J.; Mohandas, Narla; Conboy, John G.

    2003-03-25

    Among the alternative pre-mRNA splicing events that characterize protein 4.1R gene expression, one involving exon 2' plays a critical role in regulating translation initiation and N-terminal protein structure. Exon 2' encompasses translation initiation site AUG1 and is located between alternative splice acceptor sites at the 5' end of exon 2; its inclusion or exclusion from mature 4.1R mRNA regulates expression of longer or shorter isoforms of 4.1R protein, respectively. The current study reports unexpected complexity in the 5' region of the 4.1R gene that directly affects alternative splicing of exon 2'. Three mutually exclusive alternative 5' exons, designated 1A, 1B, and 1C, were identified far upstream of exon 2 in both mouse and human genomes; all three are associated with strong transcriptional promoters in the flanking genomic sequence. Importantly, exons 1A and 1B splice differentially with respect to exon 2', generating transcripts with different 5' ends and distinct N-terminal protein coding capacity. Exon 1A-type transcripts splice so as to exclude exon 2' and therefore utilize the downstream AUG2 for translation of 80kD 4.1R protein, whereas exon 1B transcripts include exon 2' and initiate at AUG1 to synthesize 135kD isoforms. RNA blot analyses revealed that 1A transcripts increase in abundance in late erythroblasts, consistent with the previously demonstrated upregulation of 80kD 4.1R during terminal erythroid differentiation. Together these results suggest that synthesis of structurally distinct 4.1R protein isoforms in various cell types is regulated by a novel mechanism requiring coordination between upstream transcription initiation events and downstream alternative splicing events.

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

    PubMed Central

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

    2015-01-01

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

  15. Changes in the expression of the human adenine nucleotide translocase isoforms condition cellular metabolic/proliferative status

    PubMed Central

    Mampel, Teresa; Viñas, Octavi

    2016-01-01

    Human cells express four mitochondrial adenine nucleotide translocase (hANT) isoforms that are tissue-specific and developmentally regulated. hANT1 is mainly expressed in terminally differentiated muscle cells; hANT2 is growth-regulated and is upregulated in highly glycolytic and proliferative cells; and hANT3 is considered to be ubiquitous and non-specifically regulated. Here, we studied how the expression of hANT isoforms is regulated by proliferation and in response to metabolic stimuli, and examined the metabolic consequences of their silencing and overexpression. In HeLa and HepG2 cells, expression of hANT3 was upregulated by shifting metabolism towards oxidation or by slowed growth associated with contact inhibition or growth-factor deprivation, indicating that hANT3 expression is highly regulated. Under these conditions, changes in hANT2 mRNA expression were not observed in either HeLa or HepG2 cells, whereas in SGBS preadipocytes (which, unlike HeLa and HepG2 cells, are growth-arrest-sensitive cells), hANT2 mRNA levels decreased. Additionally, overexpression of hANT2 promoted cell growth and glycolysis, whereas silencing of hANT3 decreased cellular ATP levels, limited cell growth and induced a stress-like response. Thus, cancer cells require both hANT2 and hANT3, depending on their proliferation status: hANT2 when proliferation rates are high, and hANT3 when proliferation slows. PMID:26842067

  16. Possible expression of a particular gamma-aminobutyric acid transporter isoform responsive to upregulation by hyperosmolarity in rat calvarial osteoblasts.

    PubMed

    Fujimori, Sayumi; Hinoi, Eiichi; Takarada, Takeshi; Iemata, Mika; Takahata, Yoshifumi; Yoneda, Yukio

    2006-11-21

    Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter in the brain, but widely distributed in different peripheral organs. We have previously shown the functional expression of GABA(B) receptors required for GABAergic signal input by cultured rat calvarial osteoblasts. This study focused on the possible functional expression of the machinery required for GABAergic signal termination such as GABA transporters. In rat calvarial osteoblasts cultured for 7 days, [(3)H]GABA accumulation was observed in a temperature-, sodium- and chloride-dependent manner, consisting of a single component with a K(m) value of 789.6+/-9.0 microM and a V(max) value of 4.4+/-0.1 nmol/min/mg protein, respectively. Both nipecotic and L-2,4-diaminobutyric acids significantly inhibited [(3)H]GABA accumulation in a concentration-dependent manner. Constitutive expression was seen with mRNA for the betaine/GABA transporter-1 (BGT-1) and taurine transporter (TauT), while hyperosmotic cultivation led to significant increases in both [(3)H]GABA accumulation and BGT-1 mRNA expression without affecting TauT mRNA expression. Highly immunoreactive cells were detected for the BGT-1 isoform at the surface of trabecular bone of neonatal rat tibias. Sustained exposure to GABA significantly inhibited alkaline phosphatase (ALP) activity, but not cellular viability, at concentrations above 0.1 mM in osteoblasts cultured for 3 to 28 days. Nipecotic acid not only decreased ALP activity alone, but also further decreased ALP activity in osteoblasts cultured in the presence of GABA. These results suggest that the BGT-1 isoform may be functionally expressed by rat calvarial osteoblasts to play a hitherto unidentified role in mechanisms underlying hyperosmotic regulation of osteoblastogenesis.

  17. Genomic organisation of the mouse gene encoding endothelin-converting enzyme-1 (ECE-1) and mRNA expression of ECE-1 isoforms in murine tissues.

    PubMed

    Lindenau, Steffi; von Langsdorff, Christian; Saxena, Amit; Paul, Martin; Orzechowski, Hans-Dieter

    2006-05-24

    Mouse knockout-models have previously revealed important biological functions of endothelin-converting enzyme-1 (ECE-1) in normal cardiac and craniofacial development. Since human ECE-1 is expressed in various isoforms, termed a, b, c, and d, expression of which is controlled by alternative promoters, we postulated that corresponding isoforms may also be transcribed from the murine Ece1 gene. By comparative sequence analysis using exon-specific sequences of human and rat ECE-1 we have resolved the complete exon-intron structure of the murine Ece1 locus on chromosome 4. The murine Ece1 gene comprises 23 exons distributed over 100 kb of genomic DNA and was found to be structurally highly conserved when compared to the human ECE1 gene. As with the human gene, the exons containing isoform-specific sequences were localised in the 5' terminal region of the murine Ece1 gene. Using specific sense primers, isoform-specific expression of murine ECE-1 mRNA in various mouse tissues was confirmed by RT-PCR. Using real-time PCR we demonstrated that ECE-1c was the most abundantly expressed isoform in most tissues, except for heart and aorta displaying a more even isoform distribution. We detected an additional isoform-specific exon, designated c2, which was apparently constitutively spliced and expressed only as minor fraction of ECE-1c transcripts. Our results provide evidence of structural conservation of mammalian genes encoding ECE-1 and will facilitate a more refined analysis of ECE-1 mRNA expression in the mouse model organism.

  18. Expression of mouse Fbxw7 isoforms is regulated in a cell cycle- or p53-dependent manner

    SciTech Connect

    Matsumoto, Akinobu; Onoyama, Ichiro; Nakayama, Keiichi I. . E-mail: nakayak1@bioreg.kyushu-u.ac.jp

    2006-11-10

    Fbxw7 is the F-box protein component of an SCF-type ubiquitin ligase that contributes to the ubiquitin-dependent degradation of cell cycle activators and oncoproteins. Three isoforms ({alpha}, {beta}, and {gamma}) of Fbxw7 are produced from mRNAs with distinct 5' exons. We have now investigated regulation of Fbxw7 expression in mouse tissues. Fbxw7{alpha} mRNA was present in all tissues examined, whereas Fbxw7{beta} mRNA was detected only in brain and testis, and Fbxw7{gamma} mRNA in heart and skeletal muscle. The amount of Fbxw7{alpha} mRNA was high during quiescence (G phase) in mouse embryonic fibroblasts (MEFs) and T cells, but it decreased markedly as these cells entered the cell cycle. The abundance of Fbxw7{alpha} mRNA was unaffected by cell irradiation or p53 status. In contrast, X-irradiation increased the amount of Fbxw7{beta} mRNA in wild-type MEFs but not in those from p53-deficient mice, suggesting that radiation-induced up-regulation of p53 leads to production of Fbxw7{beta} mRNA. Our results thus indicate that expression of Fbxw7 isoforms is differentially regulated in a cell cycle- or p53-dependent manner.

  19. Role of nuclear progesterone receptor isoforms in uterine pathophysiology

    PubMed Central

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

    2015-01-01

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

  20. Human bronchial smooth muscle cells express adenylyl cyclase isoforms 2, 4, and 6 in distinct membrane microdomains.

    PubMed

    Bogard, Amy S; Xu, Congfeng; Ostrom, Rennolds S

    2011-04-01

    Adenylyl cyclases (AC) are important regulators of airway smooth muscle function, because β-adrenergic receptor (AR) agonists stimulate AC activity and increase airway diameter. We assessed expression of AC isoforms in human bronchial smooth muscle cells (hBSMC). Reverse transcriptase-polymerase chain reaction and immunoblot analyses detected expression of AC2, AC4, and AC6. Forskolin-stimulated AC activity in membranes from hBSMC displayed Ca(2+)-inhibited and G(βγ)-stimulated AC activity, consistent with expression of AC6, AC2, and AC4. Isoproterenol-stimulated AC activity was inhibited by Ca(2+) but unaltered by G(βγ), whereas butaprost-stimulated AC activity was stimulated by G(βγ) but unaffected by Ca(2+) addition. Using sucrose density centrifugation to isolate lipid raft fractions, we found that only AC6 localized in lipid raft fractions, whereas AC2 and AC4 localized in nonraft fractions. Immunoisolation of caveolae using caveolin-1 antibodies yielded Ca(2+)-inhibited AC activity (consistent with AC6 expression), whereas the nonprecipitated material displayed G(βγ)-stimulated AC activity (consistent with expression of AC2 and/or AC4). Overexpression of AC6 enhanced cAMP production in response to isoproterenol and beraprost but did not increase responses to prostaglandin E(2) or butaprost. β(2)AR, but not prostanoid EP(2) or EP(4) receptors, colocalized with AC5/6 in lipid raft fractions. Thus, particular G protein-coupled receptors couple to discreet AC isoforms based, in part, on their colocalization in membrane microdomains. These different cAMP signaling compartments in airway smooth muscle cells are responsive to different hormones and neurotransmitters and can be regulated by different coincident signals such as Ca(2+) and G(βγ).

  1. Alternative splicing in the fiddler crab cognate ecdysteroid receptor: variation in receptor isoform expression and DNA binding properties in response to hormone.

    PubMed

    Durica, David S; Das, Sunetra; Najar, Fares; Roe, Bruce; Phillips, Barret; Kappalli, Sudha; Anilkumar, Gopinathan

    2014-09-15

    RXR cDNA cloning from three Uca species led to the identification of 4 conserved isoforms, indicative of alternative splicing in the hinge and ligand binding domains (LBD). Sequencing of overlapping clones from a Ucapugilator genomic library identified EcR isoforms matching previously identified cDNA variants; in addition, a cryptic exon in the LBD was detected and evidence for expression of this new isoform was obtained from next-generation sequencing. RNA-seq analysis also identified a new amino terminal EcR variant. EcR and RXR transcript abundance increases throughout ovarian maturation in U. pugilator, while cognate receptor transcript abundance remains constant in a related Indo-Pacific species with a different reproductive strategy. To examine if crab RXR LBD isoforms have different physical properties in vitro, electromobility shift assays were performed with different EcR isoforms. The cognate crab and fruit fly receptors differ in their responses to hormone. Ecdysteroids did not increase DNA binding for the crab heterodimers, while ecdysteroids stimulate binding for Drosophilamelanogaster EcR/USP heterodimers. In swapping experiments, UpEcR/USP heterodimers did not show ligand-responsive differences in DNA binding; both crab RXR LBD isoforms, however, conferred ligand-responsive increases in DNA binding with DmEcRs. These data indicate that both UpRXR LBD isoforms can heterodimerize with the heterologous DmEcR receptors and promote ligand and DNA binding. Unresponsiveness of the cognate receptors to ecdysteroid, however, suggest additional factors may be required to mediate endogenous, perhaps isoform-specific, differences in EcR conformation, consistent with previously reported effects of UpRXR isoforms on UpEcR ligand-binding affinities.

  2. Ouabain interactions with the α4 isoform of the sodium pump trigger non-classical steroid hormone signaling and integrin expression in spermatogenic cells.

    PubMed

    Upmanyu, Neha; Dietze, Raimund; Kirch, Ulrike; Scheiner-Bobis, Georgios

    2016-11-01

    In addition to the ubiquitous α1 isoform of the sodium pump, sperm cells also express a male-specific α4 isoform whose function has been associated with sperm motility, fertility, and capacitation. Here we investigate in the murine spermatogenic cell line GC-2 interactions of the α4 isoform with the cardiotonic steroid ouabain in signaling cascades involved in the non-classical action of steroid hormones. Exposure of GC-2 cells to low concentrations of ouabain stimulates the phosphorylation of Erk1/2 and of the transcription factors CREB and ATF-1. As a consequence of this signaling cascade, ouabain stimulates on the mRNA level the expression of integrins αv, β3 and α5, whose expression is also modulated by the cAMP response element. Increased expression of integrins αv and β3 is also seen in cultures of seminiferous tubules exposed to 10nM ouabain. At the protein level we observed a significant stimulation of β3 integrin expression by ouabain. Abrogation of α4 isoform expression by siRNA leads to the complete suppression of all ouabain-induced signaling mentioned above, including its stimulatory effect on the expression of β3 integrin. The results presented here demonstrate for the first time the induction of signaling cascades through the interaction of ouabain with the α4 isoform in a germ-cell derived cell line. The novel finding that these interactions lead to increased expression of integrins in GC-2 cells and the confirmation of these results in the ex vivo experiments indicate that hormone/receptor-like interactions of ouabain with the α4 isoform might be of significance for male physiology.

  3. Channel catfish (Ictalurus punctatus) leukocytes express estrogen receptor isoforms ERα and ERβ2 and are functionally modulated by estrogens

    USGS Publications Warehouse

    Iwanowicz, Luke R.; Stafford, James L.; Patiño, Reynaldo; Bengten, Eva; Miller, Norman W.; Blazer, Vicki

    2014-01-01

    Estrogens are recognized as modulators of immune responses in mammals and teleosts. While it is known that the effects of estrogens are mediated via leukocyte-specific estrogen receptors (ERs) in humans and mice, leucocyte-specific estrogen receptor expression and the effects of estrogens on this cell population is less explored and poorly understood in teleosts. Here in, we verify that channel catfish (Ictalurus punctaus) leukocytes express ERα and ERβ2. Transcripts of these isoforms were detected in tissue-associated leukocyte populations by PCR, but ERβ2 was rarely detected in PBLs. Expression of these receptors was temporally regulated in PBLs following polyclonal activation by concanavalin A, lipopolysaccharide or alloantigen based on evaluation by quantitative and end-point PCR. Examination of long-term leukocyte cell lines demonstrated that these receptors are differentially expressed depending on leukocyte lineage and phenotype. Expression of ERs was also temporally dynamic in some leukocyte lineages and may reflect stage of cell maturity. Estrogens affect the responsiveness of channel catfish peripheral blood leukocytes (PBLs) to mitogens in vitro. Similarly, bactericidal activity and phorbol 12-myristate 13-acetate induced respiratory burst was modulated by 17β-estradiol. These actions were blocked by the pure ER antagonist ICI 182780 indicating that response is, in part, mediated via ERα. In summary, estrogen receptors are expressed in channel catfish leukocytes and participate in the regulation of the immune response. This is the first time leukocyte lineage expression has been reported in teleost cell lines.

  4. Expression of three isoforms of Na-K-2Cl cotransporter (NKCC2) in the kidney and regulation by dehydration.

    PubMed

    Itoh, Kazuko; Izumi, Yuichiro; Inoue, Takeaki; Inoue, Hideki; Nakayama, Yushi; Uematsu, Takayuki; Fukuyama, Takashi; Yamazaki, Taiga; Yasuoka, Yukiko; Makino, Takeshi; Nagaba, Yasushi; Tomita, Kimio; Kobayashi, Noritada; Kawahara, Katsumasa; Mukoyama, Masashi; Nonoguchi, Hiroshi

    2014-10-24

    Sodium reabsorption via Na-K-2Cl cotransporter 2 (NKCC2) in the thick ascending limbs has a major role for medullary osmotic gradient and subsequent water reabsorption in the collecting ducts. We investigated intrarenal localization of three isoforms of NKCC2 mRNA expressions and the effects of dehydration on them in rats. To further examine the mechanisms of dehydration, the effects of hyperosmolality on NKCC2 mRNA expression in microdissected renal tubules was studied. RT-PCR and RT-competitive PCR were employed. The expressions of NKCC2a and b mRNA were observed in the cortical thick ascending limbs (CAL) and the distal convoluted tubules (DCT) but not in the medullary thick ascending limbs (MAL), whereas NKCC2f mRNA expression was seen in MAL and CAL. Two-day dehydration did not affect these mRNA expressions. In contrast, hyperosmolality increased NKCC2 mRNA expression in MAL in vitro. Bradykinin dose-dependently decreased NKCC2 mRNA expression in MAL. However, dehydration did not change NKCC2 protein expression in membrane fraction from cortex and outer medulla and in microdissected MAL. These data show that NKCC2a/b and f types are mainly present in CAL and MAL, respectively. Although NKCC2 mRNA expression was stimulated by hyperosmolality in vitro, NKCC2 mRNA and protein expressions were not stimulated by dehydration in vivo. These data suggest the presence of the inhibitory factors for NKCC2 expression in dehydration. Considering the role of NKCC2 for the countercurrent multiplier system, NKCC2f expressed in MAL might be more important than NKCC2a/b.

  5. Mining expression and prognosis of topoisomerase isoforms in non-small-cell lung cancer by using Oncomine and Kaplan–Meier plotter

    PubMed Central

    Hou, Guo-Xin; Liu, Panpan; Yang, Jing; Wen, Shijun

    2017-01-01

    DNA topoisomerases are essential to modulate DNA topology during various cellular genetic processes. The expression and distinct prognostic value of topoisomerase isoforms in non-small-cell lung cancer (NSCLC) is not well established. In the current study, we have examined the mRNA expression of topoisomerase isoforms by using Oncomine analysis and investigated their prognostic value via the Kaplan–Meier plotter database in NSCLC patients. Our analysis indicated that the expression level of topoisomerases in lung cancer was higher compared with normal tissues. Especially, high expression of two topoisomerase isoforms, TOP2A and TOP3A, was found to be correlated to worse overall survival (OS) in all NSCLC and lung adenocarcinoma (Ade) patients, but not in lung squamous cell carcinoma (SCC) patients. In a contrast, high expression of isoforms TOP1 and TOP2B indicated better OS in all NSCLC and Ade, but not in SCC patients. Meanwhile, high expression of TOP1MT and TOP3B was not correlated with OS in NSCLC patients. Furthermore, we also demonstrated a relationship between topoisomerase isoforms and the clinicopathological features for the NSCLC patients, such as grades, clinical stages, lymph node status, smoking status, gender, chemotherapy and radiotherapy. These results support that TOP2A and TOP3A are associated with worse prognosis in NSCLC patients. In addition, our study also shows that TOP1 and TOP2B contribute to favorable prognosis in NSCLC patients. The exact prognostic significance of TOP1MT and TOP3B need to be further elucidated. Comprehensive evaluation of expression and prognosis of topoisomerase isoforms will be a benefit for the better understanding of heterogeneity and complexity in the molecular biology of NSCLC, paving a way for more accurate prediction of prognosis and discovery of potential drug targets for NSCLC patients. PMID:28355294

  6. Expression and 1,4-dihydropyridine-binding properties of brain L-type calcium channel isoforms.

    PubMed

    Sinnegger-Brauns, Martina J; Huber, Irene G; Koschak, Alexandra; Wild, Claudia; Obermair, Gerald J; Einzinger, Ursula; Hoda, Jean-Charles; Sartori, Simone B; Striessnig, Jörg

    2009-02-01

    The L-type calcium channel (LTCC) isoforms Ca(v)1.2 and Ca(v)1.3 display similar 1,4-dihydropyridine (DHP) binding properties and are both expressed in mammalian brain. Recent work implicates Ca(v)1.3 channels as interesting drug targets, but no isoform-selective modulators exist. It is also unknown to what extent Ca(v)1.1 and Ca(v)1.4 contribute to L-type-specific DHP binding activity in brain. To address this question and to determine whether DHPs can discriminate between Ca(v)1.2 and Ca(v)1.3 binding pockets, we combined radioreceptor assays and quantitative polymerase chain reaction (qPCR). We bred double mutants (Ca(v)-DM) from mice expressing mutant Ca(v)1.2 channels [Ca(v)1.2DHP(-/-)] lacking high affinity for DHPs and from Ca(v)1.3 knockouts [Ca(v)1.3(-/-)]. (+)-[(3)H]isradipine binding to Ca(v)1.2DHP(-/-) and Ca(v)-DM brains was reduced to 15.1 and 4.4% of wild type, respectively, indicating that Ca(v)1.3 accounts for 10.7% of brain LTCCs. qPCR revealed that Ca(v)1.1 and Ca(v)1.4 alpha(1) subunits comprised 0.08% of the LTCC transcripts in mouse whole brain, suggesting that they cannot account for the residual binding. Instead, this could be explained by low-affinity binding (127-fold K(d) increase) to the mutated Ca(v)1.2 channels. Inhibition of (+)-[(3)H]isradipine binding to Ca(v)1.2DHP(-/-) (predominantly Ca(v)1.3) and wild-type (predominantly Ca(v)1.2) brain membranes by unlabeled DHPs revealed a 3- to 4-fold selectivity of nitrendipine and nifedipine for the Ca(v)1.2 binding pocket, a finding further confirmed with heterologously expressed channels. This suggests that small differences in their binding pockets may allow development of isoform-selective modulators for LTCCs and that, because of their very low expression, Ca(v)1.1 and Ca(v)1.4 are unlikely to serve as drug targets to treat CNS diseases.

  7. Differential expression of functional guanylyl cyclases in melanocytes: absence of nitric-oxide-sensitive isoform in metastatic cells.

    PubMed

    Ivanova, K; Das, P K; van den Wijngaard, R M; Lenz, W; Klockenbring, T; Malcharzyk, V; Drummer, C; Gerzer, R

    2001-03-01

    Nitric oxide (NO) is a reactive endogenous molecule with multiple functions and its cellular signaling activity is mainly mediated by activation of the soluble isoform of guanylyl cyclase, a heterodimeric (alpha/beta) hemeprotein. The expression of the NO-sensitive soluble isoform of guanylyl cyclase was studied in various cultured melanocytic cells by measuring the accumulation of guanosine 3',5'-cyclic monophosphate in the presence and absence of NO donors. Here we report that 3-morpholino-sydnonimine, a donor of NO redox species, and (Z)-1-[2- (2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate, a direct NO donor, induced a 20-fold increase in intracellular guanosine 3',5'-cyclic monophosphate in nonmetastatic melanoma cells and normal melanocytes in culture that could be related to cellular melanin content in a concentration-dependent manner. The increased intracellular guanosine 3',5'-cyclic monophosphate was due to stimulation of the activity of soluble guanylyl cyclase as such increase was completely abolished by using a specific inhibitor of soluble guanylyl cyclase. The involvement of functional soluble guanylyl cyclase was further confirmed by the presence of alpha1 and beta1 subunits in these cells at both mRNA and protein levels. In contrast, none of the NO donors induced guanosine 3',5'-cyclic monophosphate production in metastatic melanoma cells, which could be attributed to the absence of the beta1 subunit that is essential for catalytic activity of the soluble isoform of guanylyl cyclase. Metastatic melanoma cells produced higher levels of intracellular guanosine 3',5'-cyclic monophosphate in response to natriuretic peptides than other cell types, however, due to upregulation of membrane-bound guanylyl cyclase activities, but they are less pigmented or unpigmented. The present finding suggests that NO signaling in association with melanogenesis is dependent on the soluble isoform of guanylyl cyclase, whereas absence of soluble guanylyl

  8. The mitochondrial aspartate/glutamate carrier isoform 1 gene expression is regulated by CREB in neuronal cells

    PubMed Central

    Menga, Alessio; Iacobazzi, Vito; Infantino, Vittoria; Avantaggiati, Maria Laura; Palmieri, Ferdinando

    2015-01-01

    The aspartate/glutamate carrier isoform 1 is an essential mitochondrial transporter that exchanges intramitochondrial aspartate and cytosolic glutamate across the inner mitochondrial membrane. It is expressed in brain, heart and muscle and is involved in important biological processes, including myelination. However, the signals that regulate the expression of this transporter are still largely unknown. In this study we first identify a CREB binding site within the aspartate/glutamate carrier gene promoter that acts as a strong enhancer element in neuronal SH-SY5Y cells. This element is regulated by active, phosphorylated CREB protein and by signal pathways that modify the activity of CREB itself and, most noticeably, by intracellular Ca2+ levels. Specifically, aspartate/glutamate carrier gene expression is induced via CREB by forskolin while it is inhibited by the PKA inhibitor, H89. Furthermore, the CREB-induced activation of gene expression is increased by thapsigargin, which enhances cytosolic Ca2+, while it is inhibited by BAPTA-AM that reduces cytosolic Ca2+ or by STO-609, which inhibits CaMK-IV phosphorylation. We further show that CREB-dependent regulation of aspartate/glutamate carrier gene expression occurs in neuronal cells in response to pathological (inflammation) and physiological (differentiation) conditions. Since this carrier is necessary for neuronal functions and is involved in myelinogenesis, our results highlight that targeting of CREB activity and Ca2+ might be therapeutically exploited to increase aspartate/glutamate carrier gene expression in neurodegenerative diseases. PMID:25597433

  9. Differences in type II, IV, V and VI adenylyl cyclase isoform expression between rat preadipocytes and adipocytes.

    PubMed

    Serazin-Leroy, V; Morot, M; de Mazancourt, P; Giudicelli, Y

    2001-11-26

    Adenylyl cyclase catalytic activity is low in preadipocyte membranes when compared to adipocytes. Under conditions promoting inhibition of adipocyte adenylyl cyclase activity by Gpp(NH)p, a stable GTP analog, a paradoxical increase in preadipocyte adenylyl cyclase activity was obtained. In order to explain this contradiction, expression of types II, IV, V and VI adenylyl cyclase isoforms was compared in adipocytes and undifferentiated preadipocytes both by western blots and by a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) assay. Type II, IV, V and VI mRNAs and proteins were present in both adipocytes and preadipocytes. However, in undifferentiated preadipocytes, expression of type II mRNA and protein were significantly higher whereas expression of type IV, V and VI adenylyl cyclase mRNAs and proteins were significantly weaker than in adipocytes. In late differentiated preadipocytes, the adenylyl cyclase subtype mRNA expression pattern was intermediary between the undifferentiated and the full differentiation states except for type IV which remained weakly expressed. Moreover, one of the representative regulators of G-protein signaling (RGS protein), RGS4, was less expressed in undifferentiated preadipocyte membranes and cytosol extracts, which contrasts with adipocytes where RGS4 is clearly expressed. Thus, the preferential expression of type II adenylyl cyclase (G(betagamma) subunit-stimulated) in preadipocytes might explain why Gpp(NH)p elicits stimulation of adenylyl cyclase under conditions designed to promote inhibition. Conversely, the preferential expression of type V and VI adenylyl cyclases and the slightly higher expression of type IV adenylyl cyclase in adipocytes could contribute to explain the elevated total catalytic activity observed in mature fat cells compared to their precursor cells.

  10. Nonmuscle and muscle tropomyosin isoforms are expressed from a single gene by alternative RNA splicing and polyadenylation.

    PubMed Central

    Helfman, D M; Cheley, S; Kuismanen, E; Finn, L A; Yamawaki-Kataoka, Y

    1986-01-01

    The molecular basis for the expression of rat embryonic fibroblast tropomyosin 1 and skeletal muscle beta-tropomyosin was determined. cDNA clones encoding these tropomyosin isoforms exhibit complete identity except for two carboxy-proximal regions (amino acids 189 to 213 and 258 to 284) and different 3'-untranslated sequences. The isoform-specific regions delineate the troponin T-binding domains of skeletal muscle tropomyosin. Analysis of genomic clones indicates that there are two separate loci in the rat genome that contain sequences complementary to these mRNAs. One locus is a pseudogene. The other locus contains a single gene made up of 11 exons and spans approximately 10 kilobases. Sequences common to all mRNAs were found in exons 1 through 5 (amino acids 1 to 188) and exons 8 and 9 (amino acids 214 to 257). Exons 6 and 11 are specific for fibroblast mRNA (amino acids 189 to 213 and 258 to 284, respectively), while exons 7 and 10 are specific for skeletal muscle mRNA (amino acids 189 to 213 and 258 to 284, respectively). In addition, exons 10 and 11 each contain the entire 3'-untranslated sequences of the respective mRNAs including the polyadenylation site. Although the gene is also expressed in smooth muscle (stomach, uterus, and vas deferens), only the fibroblast-type splice products can be detected in these tissues. S1 and primer extension analyses indicate that all mRNAs expressed from this gene are transcribed from a single promoter. The promoter was found to contain G-C-rich sequences, a TATA-like sequence TTTTA, no identifiable CCAAT box, and two putative Sp1-binding sites. Images PMID:2432392

  11. A Switch in Akt Isoforms Is Required for Notch-Induced Snail1 Expression and Protection from Cell Death

    PubMed Central

    Frías, Alex; Lambies, Guillem; Viñas-Castells, Rosa; Martínez-Guillamon, Catalina; Dave, Natàlia

    2015-01-01

    Notch activation in aortic endothelial cells (ECs) takes place at embryonic stages during cardiac valve formation and induces endothelial-to-mesenchymal transition (EndMT). Using aortic ECs, we show here that active Notch expression promotes EndMT, resulting in downregulation of vascular endothelial cadherin (VE-cadherin) and upregulation of mesenchymal genes such as those for fibronectin and Snail1/2. In these cells, transforming growth factor β1 exacerbates Notch effects by increasing Snail1 and fibronectin activation. When Notch-downstream pathways were analyzed, we detected an increase in glycogen synthase kinase 3β (GSK-3β) phosphorylation and inactivation that facilitates Snail1 nuclear retention and protein stabilization. However, the total activity of Akt was downregulated. The discrepancy between Akt activity and GSK-3β phosphorylation is explained by a Notch-induced switch in the Akt isoforms, whereby Akt1, the predominant isoform expressed in ECs, is decreased and Akt2 transcription is upregulated. Mechanistically, Akt2 induction requires the stimulation of the β-catenin/TCF4 transcriptional complex, which activates the Akt2 promoter. Active, phosphorylated Akt2 translocates to the nucleus in Notch-expressing cells, resulting in GSK-3β inactivation in this compartment. Akt2, but not Akt1, colocalizes in the nucleus with lamin B in the nuclear envelope. In addition to promoting GSK-3β inactivation, Notch downregulates Forkhead box O1 (FoxO1), another Akt2 nuclear substrate. Moreover, Notch protects ECs from oxidative stress-induced apoptosis through an Akt2- and Snail1-dependent mechanism. PMID:26711268

  12. Human thyroid peroxidase (TPO) isoforms, TPO-1 and TPO-2: analysis of protein expression in Graves' thyroid tissue.

    PubMed

    Gardas, A; Lewartowska, A; Sutton, B J; Pasieka, Z; McGregor, A M; Banga, J P

    1997-11-01

    Thyroid peroxidase (TPO) is the key enzyme involved in the biosynthesis of thyroid hormones and is an important autoantigen in autoimmune thyroid disease. Different messenger RNA species coding for TPO are present in thyroid tissue, including the species coding for a 933-amino acid protein (termed TPO-1) and a second in which exon 10 is deleted and which is 57 residues shorter (termed TPO-2). However, it is not known whether the smaller, TPO-2 isoform is expressed as a protein in thyroid cells. In SDS-PAGE under reducing conditions, TPO appears in the thyroid microsome and purified protein preparations as a closely migrating double band of approximately 105 (larger form) and 100 kilodaltons (smaller form). We investigated the presence of the isoform TPO-2 polypeptide in Graves' thyroid tissue using rabbit antisera to three different synthetic peptides from exon 10 (specific for TPO-1) and a polyclonal rabbit and monoclonal anti-TPO antibody (both of which are specific for the two forms of TPO). The larger and smaller forms of TPO were purified by electroelution after gel electrophoresis of highly purified natural TPO from Graves' thyroid microsomes. Both of the purified forms of TPO react with all three anti-exon 10 peptide antibodies, the polyclonal anti-TPO and the monoclonal antibody anti-TPO. This shows that both forms of TPO contain exon 10-encoded polypeptide of TPO-1. Interestingly, the proportion of the larger and smaller forms of TPO varied in different Graves' thyroid microsome preparations. To investigate the presence of the smaller TPO-2 isoform in the purified natural TPO preparation, affinity depletion of TPO-1 using the anti-exon 10 peptide antibodies was carried out. The binding of anti-exon 10 peptide antibodies to the immunodepleted TPO-1 fraction was considerably diminished in comparison to binding of polyclonal anti-TPO, suggesting the presence of small amounts (< 10%) of TPO-2 expressed as a protein in thyroid cells. Our results extend previous

  13. Cardiac glycoside ouabain induces activation of ATF-1 and StAR expression by interacting with the α4 isoform of the sodium pump in Sertoli cells.

    PubMed

    Dietze, Raimund; Konrad, Lutz; Shihan, Mazen; Kirch, Ulrike; Scheiner-Bobis, Georgios

    2013-03-01

    Sertoli cells express α1 and α4 isoforms of the catalytic subunit of Na(+),K(+)-ATPase (sodium pump). Our recent findings demonstrated that interactions of the α4 isoform with cardiotonic steroids (CTS) like ouabain induce signaling cascades that resemble the so-called non-classical testosterone pathway characterized by activation of the c-Src/c-Raf/Erk1/2/CREB signaling cascade. Here we investigate a possible physiological significance of the activated cascade. The results obtained in the current investigation show that the ouabain-induced signaling cascade also leads to the activation of the CREB-related activating transcription factor 1 (ATF-1) in the Sertoli cell line 93RS2 in a concentration- and time-dependent manner, as demonstrated by detection of ATF-1 phosphorylated on Ser63 in western blots. The ouabain-activated ATF-1 protein was found to localize to the cell nuclei. The sodium pump α4 isoform mediates this activation, as it is ablated when cells are incubated with siRNA to the α4 isoform. Ouabain also leads to increased expression of steroidogenic acute regulator (StAR) protein, which has been shown to be a downstream consequence of CREB/ATF-1 activation. Taking into consideration that CTS are most likely produced endogenously, the demonstrated induction of StAR expression by ouabain establishes a link between CTS, the α4 isoform of the sodium pump, and steroidogenesis crucial for male fertility and reproduction.

  14. Different expression patterns of renal Na(+)/K(+)-ATPase α-isoform-like proteins between tilapia and milkfish following salinity challenges.

    PubMed

    Yang, Wen-Kai; Chung, Chang-Hung; Cheng, Hui Chen; Tang, Cheng-Hao; Lee, Tsung-Han

    2016-12-01

    Euryhaline teleosts can survive in a broad range of salinity via alteration of the molecular mechanisms in certain osmoregulatory organs, including in the gill and kidney. Among these mechanisms, Na(+)/K(+)-ATPase (NKA) plays a crucial role in triggering ion-transporting systems. The switch of NKA isoforms in euryhaline fish gills substantially contributes to salinity adaptation. However, there is little information about switches in the kidneys of euryhaline teleosts. Therefore, the responses of the renal NKA α-isoform protein switch to salinity challenge in euryhaline tilapia (Oreochromis mossambicus) and milkfish (Chanos chanos) with different salinity preferences were examined and compared in this study. Immunohistochemical staining in tilapia kidneys revealed the localization of NKA in renal tubules rather than in the glomeruli, similar to our previous findings in milkfish kidneys. Protein abundance in the renal NKA pan α-subunit-like, α1-, and α3-isoform-like proteins in seawater-acclimated tilapia was significantly higher than in the freshwater group, whereas the α2-isoform-like protein exhibited the opposite pattern of expression. In the milkfish, higher protein abundance in the renal NKA pan α-subunit-like and α1-isoform-like proteins was found in freshwater-acclimated fish, whereas no difference was found in the protein abundance of α2- and α3-isoform-like proteins between groups. These findings suggested that switches for renal NKA α-isoforms, especially the α1-isoform, were involved in renal osmoregulatory mechanisms of euryhaline teleosts. Moreover, differences in regulatory responses of the renal NKA α-subunit to salinity acclimation between tilapia and milkfish revealed that divergent mechanisms for maintaining osmotic balance might be employed by euryhaline teleosts with different salinity preferences.

  15. Differential expression of RARbeta isoforms in the mouse striatum during development: a gradient of RARbeta2 expression along the rostrocaudal axis.

    PubMed

    Liao, Wen-Lin; Tsai, Hsiu-Chao; Wu, Chen-Ying; Liu, Fu-Chin

    2005-06-01

    The retinoic acid receptor RARbeta is highly expressed in the striatum of the ventral telencephalon. We studied the expression pattern of different RARbeta isoforms in the developing mouse striatum by in situ hybridization. We found a differential ontogeny of RARbeta2 and RARbeta1/3 in embryonic day (E) 13.5 lateral ganglionic eminence (striatal primordium). RARbeta2 mRNA was detected primarily in the rostral and ventromedial domains, whereas RARbeta1/3 mRNAs were enriched in the caudal and dorsolateral domains. Notably, by E16.5, a prominent decreasing gradient of RARbeta2 mRNA was present in the developing striatum along the rostrocaudal axis, i.e., RARbeta2 was expressed at higher levels in the rostral than the caudal striatum. No such gradient was found for RARbeta1/3 and RARbeta3 mRNAs. The rostrocaudal RARbeta2 gradient gradually disappeared postnatally and was absent in the adult striatum. The differential expression pattern of RARbeta isoforms in the developing striatum may provide an anatomical basis for differential gene regulation by RARbeta signaling.

  16. Elevated PrPC expression predisposes to increased HSV-1 pathogenicity.

    PubMed

    Thackray, Alana M; Bujdoso, Raymond

    2006-01-01

    PrPC is a ubiquitously expressed glycophos-phatidylinositol-linked cell-surface glycoprotein found primarily in neural tissue. Although its normal function has not been established, there is evidence suggesting that PrPC is involved in cell signalling and cellular homeostasis. This suggests that variation in neuronal expression levels of this protein contributes towards pathogenicity induced by neurotropic agents. We have investigated the pathological response to infection with herpes simplex virus type-1 (HSV-1) in strains of mice that express different levels of PrPC. Prnp-/- mice fail to express PrPC due to an interruption in the open reading frame of the Prnp gene, whilst tg19 and tga20 mice express approximately 5 and 10 times more PrPC protein, respectively, than wild-type animals. Mice that express normal or increased levels of PrPC protein were more susceptible to acute HSV-1 infection than Prnp-/- mice. Following ear pinna inoculation with HSV-1 SC16, the order of susceptibility was tga20>tg19>wild-type>Prnp-/-. This trend was reversed when latent virus was assessed. Prnp-/- mice expressed significantly higher levels of latency-associated transcript-positive neurons in various tissues when compared with wild-type, tg19 and tga20 mice. Collectively, our data show that acute HSV-1 replication proceeds more efficiently in neuronal tissue that expresses PrPC protein and lends support to the view that this protein is involved in regulation of neurotropic viral pathogenesis. This suggests that interference of PrPC expression, or possible biochemical pathways associated with its function, may serve as an effective means of limiting the pathogenesis of acute HSV-1 infection.

  17. Regional heterogeneity of cellular prion protein isoforms in the mouse brain.

    PubMed

    Beringue, Vincent; Mallinson, Gary; Kaisar, Maria; Tayebi, Mourad; Sattar, Zahid; Jackson, Graham; Anstee, David; Collinge, John; Hawke, Simon

    2003-09-01

    Prion diseases are a group of invariably fatal neurodegenerative disorders that include Creutzfeldt-Jakob disease in humans, scrapie in sheep and goats, and bovine spongiform encephalopathy in cattle. The infectious agent or prion is largely composed of an abnormal isoform (PrPSc) of a host encoded normal cellular protein (PrPc). The conversion of PrPc to PrPSc is a dynamic process and, for reasons that are not clear, the distribution of spongiform change and PrPSc deposition varies among prion strains. An obvious explanation for this would be that the transformation efficiency in any given brain region depends on favourable interactions between conformations of PrPc and the prion strain being propagated within it. However, identification of specific PrPc conformations has until now been hampered by a lack of suitable panels of antibodies that discriminate PrPc subspecies under native conditions. In this study, we show that monoclonal antibodies raised against recombinant human prion protein folded into alpha or beta conformations exhibit striking heterogeneity in their specificity for truncations and glycoforms of mouse brain PrPc. We then show that some of these PrPc isoforms are expressed differentially in certain mouse brain regions. This suggests that variation in the expression of PrPc conformations in different brain regions may dictate the pattern of PrPSc deposition and vacuolation, characteristic for different prion strains.

  18. Prion neuropathology follows the accumulation of alternate prion protein isoforms after infective titre has peaked

    PubMed Central

    Sandberg, Malin K.; Al-Doujaily, Huda; Sharps, Bernadette; De Oliveira, Michael Wiggins; Schmidt, Christian; Richard-Londt, Angela; Lyall, Sarah; Linehan, Jacqueline M.; Brandner, Sebastian; Wadsworth, Jonathan D. F.; Clarke, Anthony R.; Collinge, John

    2014-01-01

    Prions are lethal infectious agents thought to consist of multi-chain forms (PrPSc) of misfolded cellular prion protein (PrPC). Prion propagation proceeds in two distinct mechanistic phases: an exponential phase 1, which rapidly reaches a fixed level of infectivity irrespective of PrPC expression level, and a plateau (phase 2), which continues until clinical onset with duration inversely proportional to PrPC expression level. We hypothesized that neurotoxicity relates to distinct neurotoxic species produced following a pathway switch when prion levels saturate. Here we show a linear increase of proteinase K-sensitive PrP isoforms distinct from classical PrPSc at a rate proportional to PrPC concentration, commencing at the phase transition and rising until clinical onset. The unaltered level of total PrP during phase 1, when prion infectivity increases a million-fold, indicates that prions comprise a small minority of total PrP. This is consistent with PrPC concentration not being rate limiting to exponential prion propagation and neurotoxicity relating to critical concentrations of alternate PrP isoforms whose production is PrPC concentration dependent. PMID:25005024

  19. DNA signals at isoform promoters

    PubMed Central

    Dai, Zhiming; Xiong, Yuanyan; Dai, Xianhua

    2016-01-01

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

  20. Sustained expression of a neuron-specific isoform of the Taf1 gene in development stages and aging in mice

    SciTech Connect

    Jambaldorj, Jamiyansuren; Makino, Satoshi; Munkhbat, Batmunkh; Tamiya, Gen

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer We identified the mouse homologue of neuron-specific TAF1 (N-Taf1). Black-Right-Pointing-Pointer Taf1 mRNA was expressed in most tissues and cell lines. Black-Right-Pointing-Pointer N-Taf1 mRNA was expressed in the brain and Neuroblastoma N2a cell lines. Black-Right-Pointing-Pointer Taf1 and N-Taf1 showed different expression profile in development stage and aging. -- Abstract: TATA-box binding protein associated factor 1 (TAF1) protein is the largest and the essential component of the TFIID complex in the pathway of RNA polymerase II-mediated gene transcription, and it regulates transcription of a large number of genes related to cell division. The neuron-specific isoform of the TAF1 gene (N-TAF1), which we reported previously, may have an essential role in neurons through transcriptional regulation of many neuron-specific genes. In the present study, we cloned the full-length cDNA that encodes the mouse homologue of N-TAF1 (N-Taf1) protein. By carrying out of real time RT-PCR, we investigated the expression analysis of the N-Taf1 mRNA in mouse tissues and cell lines. As well as the human N-TAF1, the N-Taf1 showed limited expression in the brain and neuroblastoma, whereas Taf1 expressed elsewhere. Furthermore, in mouse embryo head or mouse brain, mRNA expression of TAF1 changes dramatically during development but N-Taf1 showed sustained expression. Our result suggests that the N-Taf1 gene has an important role in non-dividing neuronal cell rather than in cell division and proliferation during neurogenesis.

  1. Differential Expression of Melanopsin Isoforms Opn4L and Opn4S during Postnatal Development of the Mouse Retina

    PubMed Central

    Hughes, Steven; Welsh, Laura; Katti, Christiana; González-Menéndez, Irene; Turton, Michael; Halford, Stephanie; Sekaran, Sumathi; Peirson, Stuart N.; Hankins, Mark W.; Foster, Russell G.

    2012-01-01

    Photosensitive retinal ganglion cells (pRGCs) respond to light from birth and represent the earliest known light detection system to develop in the mouse retina. A number of morphologically and functionally distinct subtypes of pRGCs have been described in the adult retina, and have been linked to different physiological roles. We have previously identified two distinct isoforms of mouse melanopsin, Opn4L and Opn4S, which are generated by alternate splicing of the Opn4 locus. These isoforms are differentially expressed in pRGC subtypes of the adult mouse retina, with both Opn4L and Opn4S detected in M1 type pRGCs, and only Opn4L detected in M2 type pRGCs. Here we investigate the developmental expression of Opn4L and Opn4S and show a differential profile of expression during postnatal development. Opn4S mRNA is detected at relatively constant levels throughout postnatal development, with levels of Opn4S protein showing a marked increase between P0 and P3, and then increasing progressively over time until adult levels are reached by P10. By contrast, levels of Opn4L mRNA and protein are low at birth and show a marked increase at P14 and P30 compared to earlier time points. We suggest that these differing profiles of expression are associated with the functional maturation of M1 and M2 subtypes of pRGCs. Based upon our data, Opn4S expressing M1 type pRGCs mature first and are the dominant pRGC subtype in the neonate retina, whereas increased expression of Opn4L and the maturation of M2 type pRGCs occurs later, between P10 and P14, at a similar time to the maturation of rod and cone photoreceptors. We suggest that the distinct functions associated with these cell types will develop at different times during postnatal development. PMID:22496826

  2. Differential expression of melanopsin isoforms Opn4L and Opn4S during postnatal development of the mouse retina.

    PubMed

    Hughes, Steven; Welsh, Laura; Katti, Christiana; González-Menéndez, Irene; Turton, Michael; Halford, Stephanie; Sekaran, Sumathi; Peirson, Stuart N; Hankins, Mark W; Foster, Russell G

    2012-01-01

    Photosensitive retinal ganglion cells (pRGCs) respond to light from birth and represent the earliest known light detection system to develop in the mouse retina. A number of morphologically and functionally distinct subtypes of pRGCs have been described in the adult retina, and have been linked to different physiological roles. We have previously identified two distinct isoforms of mouse melanopsin, Opn4L and Opn4S, which are generated by alternate splicing of the Opn4 locus. These isoforms are differentially expressed in pRGC subtypes of the adult mouse retina, with both Opn4L and Opn4S detected in M1 type pRGCs, and only Opn4L detected in M2 type pRGCs. Here we investigate the developmental expression of Opn4L and Opn4S and show a differential profile of expression during postnatal development. Opn4S mRNA is detected at relatively constant levels throughout postnatal development, with levels of Opn4S protein showing a marked increase between P0 and P3, and then increasing progressively over time until adult levels are reached by P10. By contrast, levels of Opn4L mRNA and protein are low at birth and show a marked increase at P14 and P30 compared to earlier time points. We suggest that these differing profiles of expression are associated with the functional maturation of M1 and M2 subtypes of pRGCs. Based upon our data, Opn4S expressing M1 type pRGCs mature first and are the dominant pRGC subtype in the neonate retina, whereas increased expression of Opn4L and the maturation of M2 type pRGCs occurs later, between P10 and P14, at a similar time to the maturation of rod and cone photoreceptors. We suggest that the distinct functions associated with these cell types will develop at different times during postnatal development.

  3. Ontogeny of mRNA expression and activity of long-chain acyl-CoA synthetase (ACSL) isoforms in Mus musculus heart.

    PubMed

    de Jong, Hendrik; Neal, Andrea C; Coleman, Rosalind A; Lewin, Tal M

    2007-01-01

    Long-chain acyl-CoA synthetases (ACSL) activate fatty acids (FA) and provide substrates for virtually every metabolic pathway that catabolizes FA or synthesizes complex lipids. We have hypothesized that each of the five cloned ACSL isoforms partitions FA towards specific downstream pathways. Adult heart expresses all five cloned ACSL isoforms, but their independent functional roles have not been elucidated. Studies implicate ACSL1 in both oxidative and lipid synthetic pathways. To clarify the functional role of ACSL1 and the other ACSL isoforms (3-6), we examined ACS specific activity and Acsl mRNA expression in the developing mouse heart which increases FA oxidative pathways for energy production after birth. Compared to the embryonic heart, ACS specific activity was 14-fold higher on post-natal day 1 (P1). On P1, as compared to the fetus, only Acsl1 mRNA increased, whereas transcripts for the other Acsl isoforms remained the same, suggesting that ACSL1 is the major isoform responsible for activating long-chain FA for myocardial oxidation after birth. In contrast, the mRNA abundance of Acsl3 was highest on E16, and decreased dramatically by P7, suggesting that ACSL3 may play a critical role during the development of the fetal heart. Our data support the hypothesis that each ACSL has a specific role in the channeling of FA towards distinct metabolic fates.

  4. Expression of embryonic fibronectin isoform EIIIA parallels alpha-smooth muscle actin in maturing and diseased kidney.

    PubMed

    Barnes, V L; Musa, J; Mitchell, R J; Barnes, J L

    1999-06-01

    In this study we examined if an association exists between expression of an alternatively spliced "embryonic" fibronectin isoform EIIIA (Fn-EIIIA) and alpha-smooth muscle actin (alpha-SMA) in the maturing and adult rat kidney and in two unrelated models of glomerular disease, passive accelerated anti-glomerular basement membrane (GBM) nephritis and Habu venom (HV)-induced proliferative glomerulonephritis, using immunohistochemistry and in situ hybridization. Fn-EIIIA and alpha-SMA proteins were abundantly expressed in mesangium and in periglomerular and peritubular interstitium of 20-day embryonic and 7-day (D-7) postnatal kidneys in regions of tubule and glomerular development. Staining was markedly reduced in these structures in maturing juvenile (D-14) kidney and was largely lost in adult kidney. Expression of Fn-EIIIA and alpha-SMA was reinitiated in the mesangium and the periglomerular and peritubular interstitium in both models and was also observed in glomerular crescents in anti-GBM nephritis. Increased expression of Fn-EIIIA mRNA by in situ hybridization corresponded to the localization of protein staining. Dual labeling experiments verified co-localization of Fn-EIIIA and alpha-SMA, showing a strong correlation of staining between location and staining intensity during kidney development, maturation, and disease. Expression of EIIIA mRNA corresponded to protein expression in developing and diseased kidneys and was lost in adult kidney. These studies show a recapitulation of the co-expression of Fn-EIIIA and alpha-SMA in anti-GBM disease and suggest a functional link for these two proteins.

  5. Molecular Cloning of HbPR-1 Gene from Rubber Tree, Expression of HbPR-1 Gene in Nicotiana benthamiana and Its Inhibition of Phytophthora palmivora

    PubMed Central

    Khunjan, Uraiwan; Ekchaweng, Kitiya; Panrat, Tanate; Tian, Miaoying; Churngchow, Nunta

    2016-01-01

    This is the first report to present a full-length cDNA (designated HbPR-1) encoding a putative basic HbPR-1 protein from rubber tree (Hevea brasiliensis) treated with salicylic acid. It was characterized and also expressed in Nicotiana benthamiana using Agrobacterium-mediated transient gene expression system in order to investigate the role of HbPR-1 gene in rubber tree against its oomycete pathogen Phytopthora palmivora and to produce recombinant HbPR-1 protein for microbial inhibition test. The HbPR-1 cDNA was 647 bp long and contained an open reading frame of 492 nucleotides encoding 163 amino acid residues with a predicted molecular mass of 17,681 Da and an isoelectric point (pI) of 8.56, demonstrating that HbPR-1 protein belongs to the basic PR-1 type. The predicted 3D structure of HbPR-1 was composed of four α-helices, three β-sheets, seven strands, and one junction loop. Expression and purification of recombinant HbPR-1 protein were successful using Agrobacterium-mediated transient expression and one-step of affinity chromatography. Heterologous expression of HbPR-1 in N. benthamiana reduced necrosis areas which were inoculated with P. palmivora zoospores, indicating that the expressed HbPR-1 protein played an important role in plant resistance to pathogens. The purified recombinant HbPR-1 protein was found to inhibit 64% of P. palmivora zoospore germination on a water agar plate compared with control, suggesting that it was an antimicrobial protein against P. palmivora. PMID:27337148

  6. Molecular Cloning of HbPR-1 Gene from Rubber Tree, Expression of HbPR-1 Gene in Nicotiana benthamiana and Its Inhibition of Phytophthora palmivora.

    PubMed

    Khunjan, Uraiwan; Ekchaweng, Kitiya; Panrat, Tanate; Tian, Miaoying; Churngchow, Nunta

    2016-01-01

    This is the first report to present a full-length cDNA (designated HbPR-1) encoding a putative basic HbPR-1 protein from rubber tree (Hevea brasiliensis) treated with salicylic acid. It was characterized and also expressed in Nicotiana benthamiana using Agrobacterium-mediated transient gene expression system in order to investigate the role of HbPR-1 gene in rubber tree against its oomycete pathogen Phytopthora palmivora and to produce recombinant HbPR-1 protein for microbial inhibition test. The HbPR-1 cDNA was 647 bp long and contained an open reading frame of 492 nucleotides encoding 163 amino acid residues with a predicted molecular mass of 17,681 Da and an isoelectric point (pI) of 8.56, demonstrating that HbPR-1 protein belongs to the basic PR-1 type. The predicted 3D structure of HbPR-1 was composed of four α-helices, three β-sheets, seven strands, and one junction loop. Expression and purification of recombinant HbPR-1 protein were successful using Agrobacterium-mediated transient expression and one-step of affinity chromatography. Heterologous expression of HbPR-1 in N. benthamiana reduced necrosis areas which were inoculated with P. palmivora zoospores, indicating that the expressed HbPR-1 protein played an important role in plant resistance to pathogens. The purified recombinant HbPR-1 protein was found to inhibit 64% of P. palmivora zoospore germination on a water agar plate compared with control, suggesting that it was an antimicrobial protein against P. palmivora.

  7. Close Vicinity of PrP Expressing Cells (FDC) with Noradrenergic Fibers in Healthy Sheep Spleen

    PubMed Central

    Lezmi, S.; Hunsmann, G.; Baron, T.

    2001-01-01

    In naturally and experimentally occurring scrapie in sheep, prions invade the immune system and replicate in lymphoid organs. Here we analysed immunohistochemically, in seven spleens of 6-month-old healthy sheep, the nature of the cells expressing prion protein (PrP) potentially supporting prion replication, as well as their relationship with autonomic innervation. PrP was identified using either RB1 rabbit antiserum or 4F2 monoclonal antibody directed against AA 108–123 portion of the bovine and AA 79–92 of human prion protein respectively. Using double labelling analysis, we demonstrated that PrPc is expressed by follicular dendritic cells using a specific monoclonal antibody (CNA42). We also showed the close vicinity of these PrP expressing cells with noradrenergic fibers, using a polyclonal tyrosine hydroxylase antibody. Our results may help the study of the cellular requirements for the possible neuroinvasion from the spleen. PMID:11785673

  8. Platelet APP isoform ratios in asymptomatic young adults expressing an AD-related presenilin-1 mutation.

    PubMed

    Baskin, F; Rosenberg, R N; Iyer, L; Schellenberg, G D; Hynan, L; Nee, L E

    2001-01-15

    The Alzheimer's disease (AD) related amyloid precursor protein (APP) is stored, cleaved and released similarly from neurons and from platelets. We have reported that the proportion of 120-130 to 110 kDa carboxyl-cleaved APP present in the platelets of AD patients is significantly lower than that of platelets of age-matched controls. This reduced APP isoform ratio, not seen in several other disease groups, is further reduced as the severity of AD increases. Since the neuropathology of AD is believed to begin many years before the onset of cognitive loss, we have also compared platelet APP ratios of four pre-symptomatic young adults carrying a presenilin-1 mutation to seven siblings homozygous for the normal PS-1 gene in an effort to determine whether reduced APP ratios are present before apparent cognitive loss in familial AD. Decreased platelet APP ratios were not seen in any of these subjects at this time. We will continue to monitor these subjects as they near the mean age of AD onset in these families. As the magnitude of the APP ratio reduction is proportional to the severity of cognitive loss in sporadic AD, these cognitively normal incipient AD subjects would not be expected to present significant reductions in this AD severity index at this time. Alternatively, the absence of platelet APP ratio reductions may result from a failure of platelets from familial PS-1 AD subjects to manifest altered APPs, as has been reported for PS-2 AD subjects, unlike those of sporadic AD patients. Continued monitoring of cognitive status in our sub-set of controls with AD-like low APP ratios may yet validate the ability of this assay to detect incipient sporadic AD.

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

    PubMed

    Sahu, Binod Bihari; Shaw, Birendra Prasad

    2009-07-01

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

  10. Reduced Expression of the Liver/Beta-Cell Glucose Transporter Isoform in Glucose-Insensitive Pancreatic Beta Cells of Diabetic Rats

    NASA Astrophysics Data System (ADS)

    Thorens, Bernard; Weir, Gordon C.; Leahy, John L.; Lodish, Harvey F.; Bonner-Weir, Susan

    1990-09-01

    Rats injected with a single dose of streptozocin at 2 days of age develop non-insulin-dependent diabetes 6 weeks later. The pancreatic beta islet cells of these diabetic rats display a loss of glucose-induced insulin secretion while maintaining sensitivity to other secretagogues such as arginine. We analyzed the level of expression of the liver/beta-cell glucose transporter isoform in diabetic islets by immunofluorescence staining of pancreas sections and by Western blotting of islet lysates. Islets from diabetic animals have a reduced expression of this beta-cell-specific glucose transporter isoform and the extent of reduction is correlated with the severity of hyperglycemia. In contrast, expression of this transporter isoform in liver is minimally modified by the diabetes. Thus a decreased expression of the liver/beta-cell glucose transporter isoform in beta cells is associated with the impaired glucose sensing characteristic of diabetic islets; our data suggest that this glucose transporter may be part of the beta-cell glucose sensor.

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

    EPA Science Inventory

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

  12. Progesterone regulates the expression and activity of two mouse isoforms of the glycoprotein folding sensor UDP-Glc: glycoprotein glucosyltransferase (UGGT).

    PubMed

    Prados, María B; Caramelo, Julio J; Miranda, Silvia E

    2013-12-01

    UDP-Glucose:glycoprotein glucosyltransferase (UGGT) is a central component of the endoplasmic reticulum (ER) glycoprotein-folding quality control system, which prevents the exit of partially folded species. UGGT activity can be regulated by the accumulation of misfolded proteins in the ER, a stimulus that triggers a complex signaling pathway known as unfolded protein response (UPR) which is closely associated with inflammation and disease. In this work, we investigated the effect of progesterone (P4) on the expression and activity of UGGT in a mouse hybridoma. We detected the expression of two UGGT isoforms, UGGT1 and UGGT2, and demonstrated that both isoforms are active in these cells. Interestingly, the expression of each isoform is regulated by high physiological P4 concentrations. This work provides the first evidence of a hormonal regulation of UGGT isoform expression and activity, which might influence the glycoprotein quality control mechanism. These findings could contribute to the study of pathologies triggered by the accumulation of misfolded proteins.

  13. Expression of Ik6 and Ik8 Isoforms and Their Association with Relapse and Death in Mexican Children with Acute Lymphoblastic Leukemia

    PubMed Central

    Reyes-León, Adriana; Juárez-Velázquez, Rocío; Medrano-Hernández, Alma; Cuenca-Roldán, Teresa; Salas-Labadía, Consuelo; del Pilar Navarrete-Meneses, María; Rivera-Luna, Roberto; López-Hernández, Gerardo; Paredes-Aguilera, Rogelio; Pérez-Vera, Patricia

    2015-01-01

    Expression of the 6 and 8 dominant-negative Ikaros isoforms in pediatric patients with acute lymphoblastic leukemia has been associated with a high risk of relapse and death; due to these isoforms disrupting the differentiation and proliferation of lymphoid cells. The aim of this study was to know the frequency of Ik6 and Ik8 in 113 Mexican ALL-children treated within the National Popular Medical Insurance Program to determine whether there was an association with relapse-free survival, event-free survival and overall survival, and to assess its usefulness in the initial stratification of patients. The expression of these isoforms was analyzed using specific primer sets and nested RT-PCR. The detected transcripts were classified according to the isoforms’s sizes reported. A non-expected band of 300 bp from one patient was analyzed by sequencing. Twenty-six patients expressed Ik6 and/or Ik8 and one of them expressed a variant of Ik8 denominated Ik8-deleted. Although the presence of them was not statistically associated with lower relapse free survival (p = 0.432), event free survival (p = 0.667) or overall survival (p = 0.531), inferior overall survival was observed in patients that expressed these isoforms and showed high or standard risk by age and white blood-cell count at diagnosis. Of the 26 patients Ik6+ and/or Ik8+, 14 did not present adverse events; from them 6 were exclusively Ik6+ and/or Ik8+, and 8 were positive for the other Ikaros isoforms (Ik1, Ik2, Ik5, Ik3A, Ik4, Ik4A, Ik7). In the patients studied, the expression of Ik6 and Ik8 did not constitute an independent prognostic factor for relapse or death related to disease; therefore, they could not be used in the initial risk stratification. PMID:26131904

  14. S1PR1 expression correlates with inflammatory responses to Newcastle disease virus infection.

    PubMed

    Li, Yaling; Xie, Peng; Sun, Minhua; Xiang, Bin; Kang, Yinfeng; Gao, Pei; Zhu, Wenxian; Ning, Zhangyong; Ren, Tao

    2016-01-01

    Newcastle disease virus (NDV) is the causative agent of Newcastle disease, which is characterized by inflammatory pathological changes in the organs of chickens. The inflammatory response to this disease has not been well characterized. Previous reports showed that the sphingosine-1-phosphate-1 receptor (S1PR1), a G protein-coupled receptor, is important to the activation of inflammatory responses. To understand better the viral pathogenesis and host inflammatory response, we analyzed S1PR1 expression during NDV infection. We observed a direct correlation between chicken embryo fibroblast (CEF) cellular inflammatory responses and S1PR1 expression. Virulent NDV-infected CEF cells also had elevated levels of pro-inflammatory cytokines (IL-1β, IL-6 and IL-18). When S1PR1 was inhibited by using the specific antagonist W146, pro-inflammatory cytokine production declined. Overexpression of S1PR1 resulted in increased virus-induced IL-1β production. S1PR1 expression levels did not impact significantly NDV replication. These findings highlight the important role of S1PR1 in inflammatory responses in NDV infection.

  15. Association between increased expression of endothelial isoform of nitric oxide synthase in the human fallopian tube and tubal ectopic pregnancy

    PubMed Central

    Fath Bayati, Leyla; Ghaffari Novin, Marefat; Fadaei Fathabadi, Fatemeh; Piryaei, Abbas; Heidari, Mohammad Hasan; Bandehpour, Mozhgan; Norouzian, Mohsen; Alizadeh Parhizgar, Mahdi; Shakooriyan Fard, Mahmood

    2014-01-01

    Background: Tubal ectopic pregnancy (tEP) is the most common type of extra-uterine pregnancy and the most common cause of maternal mortality. Nitric oxide (NO) is a molecule that incorporates in many physiological processes of female reproductive system. Recent studies have demonstrated the possible role of endothelial isoform of nitric oxide synthase (eNOS) enzyme in the regulation of many reproductive events that occur in the fallopian tube (FT). Objective: The aim of this study was to evaluate the expression of eNOS in the FTs of women with tEP. Materials and Methods: In this case-control study, a total number of 30FTs samples were obtained from three groups including: 10 FTs of women that bearing an EP, 10 FTs from the non-pregnant women at luteal phase of the menstrual cycle, and 10 FTs of healthy pregnant women (n=10). Samples were fixed in 10% buffered formalin and then were evaluated by immunohistochemistry. Results: Localization of eNOS was seen in secretory and ciliated luminal epithelium and vascular endothelium of all groups. However, we did not observed the expression of eNOS in smooth muscle cells of all groups. Expression of eNOS in luminal epithelium of women with EP compared to non-pregnant women at luteal phase of menstrual cycle and healthy pregnant group showed statistically significant increase (p=0.00). Significant difference in expression of eNOS was not observed in luminal epithelium of FTs of women at luteal phase compared to healthy pregnant groups (p=0.78). Conclusion: This study indicates that changes in expression of eNOS in luminal epithelium of FT may lead to development of EP. This article extracted from M.Sc. thesis. (Leyla Fath Bayati) PMID:24799858

  16. Overexpression of progesterone receptor A isoform in mice leads to endometrial hyperproliferation, hyperplasia and atypia.

    PubMed

    Fleisch, M C; Chou, Y C; Cardiff, Robert D; Asaithambi, A; Shyamala, G

    2009-04-01

    A delicate balance in estrogen and progesterone signaling through their cognate receptors is characteristic for the physiologic state of the endometrium, and a shift in receptor isotype expression can be frequently found in human endometrial pathology. In this study, using a transgenic mouse model, we examined the mechanisms whereby alterations in progesterone receptor (PR) isotype expression leads to endometrial pathology. For an experimental model, we used transgenic mice (PR-A transgenics) carrying an imbalance in the native ratio of the two PR isoforms A and B (PR-A and PR-B) through the expression of additional A form and examined their uterine phenotype under different hormonal regimens, using various criteria. Uterine epithelial cell proliferation was augmented in PR-A transgenics and was abolished by PR antagonists. In particular, proliferative response to progesterone, independent of signaling through estrogen, was enhanced. Upon continuous exposure to estradiol and progesterone, the uteri in PR-A transgenics displayed gross enlargement, endometrial hyperplasia including atypical lesions, endometritis and pelvic inflammatory disease. Imbalanced expression of the two isoforms of PR in a transgenic model reveals multiple derangements in the regulation of uterine physiology, resulting in various pathologies including hyperplasias.

  17. Recombinant bacterial expression and purification of human fragile X mental retardation protein isoform 1.

    PubMed

    Evans, Timothy L; Mihailescu, Mihaela-Rita

    2010-12-01

    The loss of expression of the fragile X mental retardation protein (FMRP) leads to fragile X syndrome. FMRP has two types of RNA binding domains, two K-homology domains and an arginine-glycine-glycine box domain, and it is proposed to act as a translation regulator of specific messenger RNA. The interest to produce sufficient quantities of pure recombinant FMRP for biochemical and biophysical studies is high. However, the recombinant bacterial expression of FMRP has had limited success, and subsequent recombinant eukaryotic and in vitro expression has also resulted in limited success. In addition, the in vitro and eukaryotic expression systems may produce FMRP which is posttranslationally modified, as phosphorylation and arginine methylation have been shown to occur on FMRP. In this study, we have successfully isolated the conditions for recombinant expression, purification and long-term storage of FMRP using Escherichia coli, with a high yield. The expression of FMRP using E. coli renders the protein devoid of the posttranslational modifications of phosphorylation and arginine methylation, allowing the study of the direct effects of these modifications individually and simultaneously. In order to assure that FMRP retained activity throughout the process, we used fluorescence spectroscopy to assay the binding activity of the FMRP arginine-glycine-glycine box for the semaphorin 3F mRNA and confirmed that FMRP remained active.

  18. Sexually Dimorphic Expression of vasa Isoforms in the Tongue Sole (Cynoglossus semilaevis)

    PubMed Central

    Wang, Zhongkai; Gao, Jinning; Song, Huayu; Wu, Xiaomeng; Sun, Yan; Qi, Jie; Yu, Haiyang; Wang, Zhigang; Zhang, Quanqi

    2014-01-01

    The vasa gene encodes an ATP-dependent RNA helicase of the DEAD box protein family that functions in a broad range of molecular events involving duplex RNA. In most species, the germline specific expression of vasa becomes a molecular marker widely used in the visualization and labeling of primordial germ cells (PGCs) and a tool in surrogate broodstock production through PGC transplantation. The vasa gene from tongue sole (Cynoglossus semilaevis) was characterized to promote the development of genetic breeding techniques in this species. Three C. semilaevis vasa transcripts were isolated, namely vas-l, vas-m, and vas-s. Quantitative real-time PCR results showed that C. semilaevis vasa transcripts were prevalently expressed in gonads, with very weak expression of vas-s in other tissues. Embryonic development expression profiles revealed the onset of zygotic transcription of vasa mRNAs and the maternal deposit of the three transcripts. The genetic ZW female juvenile fish was discriminated from genetic ZZ males by a pair of female specific primers. Only the expression of vas-s can be observed in both sexes during early gonadal differentiation. Before PGCs started mitosis, there was sexually dimorphic expression of vas-s with the ovary showing higher levels and downward trend. The results demonstrated the benefits of vasa as a germline specific marker for PGCs during embryonic development and gonadal differentiation. This study lays the groundwork for further application of C. semilaevis PGCs in fish breeding. PMID:24671276

  19. Aquaporin 4 is a Ubiquitously Expressed Isoform in the Dogfish (Squalus acanthias) Shark.

    PubMed

    Cutler, Christopher P; Maciver, Bryce; Cramb, Gordon; Zeidel, Mark

    2011-01-01

    The dogfish ortholog of aquaporin 4 (AQP4) was amplified from cDNA using degenerate PCR followed by cloning and sequencing. The complete coding region was then obtained using 5' and 3' RACE techniques. Alignment of the sequence with AQP4 amino acid sequences from other species showed that dogfish AQP4 has high levels (up to 65.3%) of homology with higher vertebrate sequences but lower levels of homology to Agnathan (38.2%) or teleost (57.5%) fish sequences. Northern blotting indicated that the dogfish mRNA was approximately 3.2 kb and was highly expressed in the rectal gland (a shark fluid secretory organ). Semi-quantitative PCR further indicates that AQP4 is ubiquitous, being expressed in all tissues measured but at low levels in certain tissues, where the level in liver > gill >  intestine. Manipulation of the external environmental salinity of groups of dogfish showed that when fish were acclimated in stages to 120% seawater (SW) or 75% SW, there was no change in AQP4 mRNA expression in either rectal gland, kidney, or esophagus/cardiac stomach. Whereas quantitative PCR experiments using the RNA samples from the same experiment, showed a significant 63.1% lower abundance of gill AQP4 mRNA expression in 120% SW-acclimated dogfish. The function of dogfish AQP4 was also determined by measuring the effect of the AQP4 expression in Xenopus laevis oocytes. Dogfish AQP4 expressing-oocytes, exhibited significantly increased osmotic water permeability (P(f)) compared to controls, and this was invariant with pH. Permeability was not significantly reduced by treatment of oocytes with mercury chloride, as is also the case with AQP4 in other species. Similarly AQP4 expressing-oocytes did not exhibit enhanced urea or glycerol permeability, which is also consistent with the water-selective property of AQP4 in other species.

  20. Aquaporin 4 is a Ubiquitously Expressed Isoform in the Dogfish (Squalus acanthias) Shark

    PubMed Central

    Cutler, Christopher P; MacIver, Bryce; Cramb, Gordon; Zeidel, Mark

    2012-01-01

    The dogfish ortholog of aquaporin 4 (AQP4) was amplified from cDNA using degenerate PCR followed by cloning and sequencing. The complete coding region was then obtained using 5′ and 3′ RACE techniques. Alignment of the sequence with AQP4 amino acid sequences from other species showed that dogfish AQP4 has high levels (up to 65.3%) of homology with higher vertebrate sequences but lower levels of homology to Agnathan (38.2%) or teleost (57.5%) fish sequences. Northern blotting indicated that the dogfish mRNA was approximately 3.2 kb and was highly expressed in the rectal gland (a shark fluid secretory organ). Semi-quantitative PCR further indicates that AQP4 is ubiquitous, being expressed in all tissues measured but at low levels in certain tissues, where the level in liver > gill >  intestine. Manipulation of the external environmental salinity of groups of dogfish showed that when fish were acclimated in stages to 120% seawater (SW) or 75% SW, there was no change in AQP4 mRNA expression in either rectal gland, kidney, or esophagus/cardiac stomach. Whereas quantitative PCR experiments using the RNA samples from the same experiment, showed a significant 63.1% lower abundance of gill AQP4 mRNA expression in 120% SW-acclimated dogfish. The function of dogfish AQP4 was also determined by measuring the effect of the AQP4 expression in Xenopus laevis oocytes. Dogfish AQP4 expressing-oocytes, exhibited significantly increased osmotic water permeability (Pf) compared to controls, and this was invariant with pH. Permeability was not significantly reduced by treatment of oocytes with mercury chloride, as is also the case with AQP4 in other species. Similarly AQP4 expressing-oocytes did not exhibit enhanced urea or glycerol permeability, which is also consistent with the water-selective property of AQP4 in other species. PMID:22291652

  1. MicroRNA-281 regulates the expression of ecdysone receptor (EcR) isoform B in the silkworm, Bombyx mori

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hundreds of Bombyx mori miRNAs had been identified in recent years, but their function in vivo remains poorly understood. The silkworm EcR gene (BmEcR) has three transcriptional isoforms, A, B1 and B2. Isoform sequences are different in the 3’UTR region of the gene, which is the case only in insects...

  2. Isoform switching of steroid receptor co-activator-1 attenuates glucocorticoid-induced anxiogenic amygdala CRH expression.

    PubMed

    Zalachoras, I; Verhoeve, S L; Toonen, L J; van Weert, L T C M; van Vlodrop, A M; Mol, I M; Meelis, W; de Kloet, E R; Meijer, O C

    2016-12-01

    Maladaptive glucocorticoid effects contribute to stress-related psychopathology. The glucocorticoid receptor (GR) that mediates many of these effects uses multiple signaling pathways. We have tested the hypothesis that manipulation of downstream factors ('coregulators') can abrogate potentially maladaptive GR-mediated effects on fear-motivated behavior that are linked to corticotropin releasing hormone (CRH). For this purpose the expression ratio of two splice variants of steroid receptor coactivator-1 (SRC-1) was altered via antisense-mediated 'exon-skipping' in the central amygdala of the mouse brain. We observed that a change in splicing towards the repressive isoform SRC-1a strongly reduced glucocorticoid-induced responsiveness of Crh mRNA expression and increased methylation of the Crh promoter. The transcriptional GR target gene Fkbp5 remained responsive to glucocorticoids, indicating gene specificity of the effect. The shift of the SRC-1 splice variants altered glucocorticoid-dependent exploratory behavior and attenuated consolidation of contextual fear memory. In conclusion, our findings demonstrate that manipulation of GR signaling pathways related to the Crh gene can selectively diminish potentially maladaptive effects of glucocorticoids.

  3. Non-raft adenylyl cyclase 2 defines a cAMP signaling compartment that selectively regulates IL-6 expression in airway smooth muscle cells: differential regulation of gene expression by AC isoforms.

    PubMed

    Bogard, Amy S; Birg, Anna V; Ostrom, Rennolds S

    2014-04-01

    Adenylyl cyclase (AC) isoforms differ in their tissue distribution, cellular localization, regulation, and protein interactions. Most cell types express multiple AC isoforms. We hypothesized that cAMP produced by different AC isoforms regulates unique cellular responses in human bronchial smooth muscle cells (BSMC). Overexpression of AC2, AC3, or AC6 had distinct effects on forskolin (Fsk)-induced expression of a number of known cAMP-responsive genes. These data show that different AC isoforms can differentially regulate gene expression. Most notable, overexpression and activation of AC2 enhanced interleukin 6 (IL-6) expression, but overexpression of AC3 or AC6 had no effect. IL-6 production by BSMC was induced by Fsk and select G protein-coupled receptor (GPCR) agonists, though IL-6 levels did not directly correlate with global cAMP levels. Treatment with PKA selective 6-Bnz-cAMP or Epac selective 8-CPT-2Me-cAMP cAMP analogs revealed a predominant role for PKA in cAMP-mediated induction of IL-6. IL-6 promoter mutations demonstrated that AP-1 and CRE transcription sites were required for Fsk to stimulate IL-6 expression. Our present study defines an AC2 cAMP signaling compartment that specifically regulates IL-6 expression in BSMC via Epac and PKA and demonstrates that other AC isoforms are excluded from this pool.

  4. Expression of NKp46 Splice Variants in Nasal Lavage Following Respiratory Viral Infection: Domain 1-Negative Isoforms Predominate and Manifest Higher Activity

    PubMed Central

    Shemer-Avni, Yonat; Kundu, Kiran; Shemesh, Avishai; Brusilovsky, Michael; Yossef, Rami; Meshesha, Mesfin; Solomon-Alemayehu, Semaria; Levin, Shai; Gershoni-Yahalom, Orly; Campbell, Kerry S.; Porgador, Angel

    2017-01-01

    The natural killer (NK) cell activating receptor NKp46/NCR1 plays a critical role in elimination of virus-infected and tumor cells. The NCR1 gene can be transcribed into five different splice variants, but the functional importance and physiological distribution of NKp46 isoforms are not yet fully understood. Here, we shed light on differential expression of NKp46 splice variants in viral respiratory tract infections and their functional difference at the cellular level. NKp46 was the most predominantly expressed natural cytotoxicity receptor in the nasal lavage of patients infected with four respiratory viruses: respiratory syncytia virus, adenovirus, human metapneumovirus, or influenza A. Expression of NKp30 was far lower and NKp44 was absent in all patients. Domain 1-negative NKp46 splice variants (i.e., NKp46 isoform d) were the predominantly expressed isoform in nasal lavage following viral infections. Using our unique anti-NKp46 mAb, D2-9A5, which recognizes the D2 extracellular domain, and a commercial anti-NKp46 mAb, 9E2, which recognizes D1 domain, allowed us to identify a small subset of NKp46 D1-negative splice variant-expressing cells within cultured human primary NK cells. This NKp46 D1-negative subset also showed higher degranulation efficiency in term of CD107a surface expression. NK-92 cell lines expressing NKp46 D1-negative and NKp46 D1-positive splice variants also showed functional differences when interacting with targets. A NKp46 D1-negative isoform-expressing NK-92 cell line showed enhanced degranulation activity. To our knowledge, we provide the first evidence showing the physiological distribution and functional importance of human NKp46 splice variants under pathological conditions. PMID:28261217

  5. Identification and expression analysis of two interleukin-23α (p19) isoforms, in rainbow trout Oncorhynchus mykiss and Atlantic salmon Salmo salar.

    PubMed

    Jiang, Yousheng; Husain, Mansourah; Qi, Zhitao; Bird, Steve; Wang, Tiehui

    2015-08-01

    Interleukin (IL)-23 is a heterodimeric IL-12 family cytokine composed of a p19 α-chain, linked to a p40 β-chain that is shared with IL-12. IL-23 is distinguished functionally from IL-12 by its ability to induce the production of IL-17, and differentiation of Th17 cells in mammals. Three isoforms of p40 (p40a, p40b and p40c) have been found in some 3R teleosts. Salmonids also possess three p40 isoforms (p40b1, p40b2 and p40c) although p40a is missing, and two copies (paralogues) of p40b are present that have presumably been retained following the 4R duplication in this fish lineage. Teleost p19 has been discovered recently in zebrafish, but to date there is limited information on expression and modulation of this molecule. In this report we have cloned two p19 paralogues (p19a and p19b) in salmonids, suggesting that a salmonid can possess six potential IL-23 isoforms. Whilst Atlantic salmon has two active p19 genes, the rainbow trout p19b gene may have been pseudogenized. The salmonid p19 translations share moderate identities (22.8-29.9%) to zebrafish and mammalian p19 molecules, but their identity was supported by structural features, a conserved 4 exon/3 intron gene organisation, and phylogenetic tree analysis. The active salmonid p19 genes are highly expressed in blood and gonad. Bacterial (Yersinia ruckeri) and viral infection in rainbow trout induces the expression of p19a, suggesting pathogen-specific induction of IL-23 isoforms. Trout p19a expression was also induced by PAMPs (poly IC and peptidoglycan) and the proinflammatory cytokine IL-1β in primary head kidney macrophages. These data may indicate diverse functional roles of trout IL-23 isoforms in regulating the immune response in fish.

  6. Expression of LeNHX isoforms in response to salt stress in salt sensitive and salt tolerant tomato species.

    PubMed

    Gálvez, Francisco Javier; Baghour, Mourad; Hao, Gangping; Cagnac, Olivier; Rodríguez-Rosales, María Pilar; Venema, Kees

    2012-02-01

    In general, wild tomato species are more salt tolerant than cultivated species, a trait that is related to enhanced Na(+) accumulation in aerial parts in the wild species, but the molecular basis for these differences is not known. Plant NHX proteins have been suggested to be important for salt tolerance by promoting accumulation of Na(+) or K(+) inside vacuoles. Therefore, differences in expression or activity of NHX proteins in tomato could be at the basis of the enhanced salt tolerance in wild tomato species. To test this hypothesis, we studied the expression level of four NHX genes in the salt sensitive cultivated species Solanum lycopersicum L. cv. Volgogradskij and the salt tolerant wild species Solanum pimpinelifolium L in response to salt stress. First, we determined that in the absence of salt stress, the RNA abundance of LeNHX2, 3 and 4 was comparable in both species, while more LeNHX1 RNA was detected in the tolerant species. LeNHX2 and LeNHX3 showed comparable expression levels and were present in all tissues, while LeNHX4 was expressed above all in stem and fruit tissues. Next, we confirmed that the wild species was more tolerant and accumulated more Na(+) in aerial parts of the plant. This correlated with the observation that salt stress induced especially the LeNHX3 and LeNHX4 isoforms in the tolerant species. These results support a role of NHX genes as determinants of salt tolerance in tomato, inducing enhanced Na(+) accumulation observed in the wild species when grown in the presence of NaCl.

  7. Expression and function of COX isoforms in renal medulla: evidence for regulation of salt sensitivity and blood pressure.

    PubMed

    Ye, Wenling; Zhang, Hui; Hillas, Elaine; Kohan, Donald E; Miller, R Lance; Nelson, Raoul D; Honeggar, Matthew; Yang, Tianxin

    2006-02-01

    Expression of cyclooxygenase (COX)-2, but not COX-1, in the renal medulla is stimulated by chronic salt loading; yet the functional implication of this phenomenon is incompletely understood. The present study examined the cellular localization and antihypertensive function of high-salt-induced COX-2 expression in the renal medulla, with a parallel assessment of the function of COX-1. COX-2 protein expression in response to high-salt loading, assessed by immunostaining, was found predominantly in inner medullary interstitial cells, whereas COX-1 protein was abundant in collecting duct (CD) and inner medullary interstitial cells and was not affected by high salt. We compared mRNA expressions of COX-1 and COX-2 in CD vs. non-CD cells isolated from aquaporin 2-green fluorescent protein transgenic mice. A low level of COX-2 mRNA, but a high level of COX-1 mRNA, as determined by real-time RT-PCR, was detected in CD compared with non-CD segments. During high-salt intake, chronic infusions of the COX-2 blocker NS-398 and the COX-1 blocker SC-560 into the renal medulla of Sprague-Dawley rats for 5 days induced approximately 30- and 15-mmHg increases in mean arterial pressure, respectively. During similar high-salt intake, COX-1 knockout mice exhibited a gradual, but significant, increase in systolic blood pressure that was associated with a marked suppression of urinary PGE2 excretion. Therefore, we conclude that the two COX isoforms in the renal medulla play a similar role in the stabilization of arterial blood pressure during salt loading.

  8. The Proportion of ALDEFLUOR-Positive Cancer Stem Cells Changes with Cell Culture Density Due to the Expression of Different ALDH Isoforms

    PubMed Central

    Opdenaker, Lynn M.; Modarai, Shirin R.; Boman, Bruce M.

    2017-01-01

    A significant number of discrepancies exist within the literature regarding ALDEFLUOR-positive stem cell populations in cell lines. We hypothesized that these inconsistencies resulted from differences in culture conditions, particularly cell density. We cultured several colon cancer cell lines (N=8) at high and low densities and found a significant decrease in ALDEFLUOR-positive cell populations at high density. However, we found no changes in the CD166-positive stem cell population, self-renewal, or cell cycle distribution of cells cultured at different densities. Interestingly, when we sorted both ALDEFLUOR positive and negative populations from the different density cultures, we identified a significant number of Aldehyde dehydrogenase (ALDH) isoforms whose expression was decreased in ALDEFLUOR-positive stem cells cultured at high density. This novel finding suggests that multiple ALDH isoforms contribute to ALDEFLUOR activity in colon cancer stem cells and decreases in ALDEFLUOR-positive stem cells at high cell density are due to decreased expression of multiple ALDH isoforms. Thus, designing therapeutics to target ALDEFLUOR-positive cancer stem cells may require inhibition of multiple ALDH isoforms.

  9. Regulation of pyruvate dehydrogenase kinase isoform 4 (PDK4) gene expression by glucocorticoids and insulin.

    PubMed

    Connaughton, Sara; Chowdhury, Farhana; Attia, Ramy R; Song, Shulan; Zhang, Yi; Elam, Marshall B; Cook, George A; Park, Edwards A

    2010-02-05

    The pyruvate dehydrogenase complex (PDC) catalyzes the conversion of pyruvate to acetyl-CoA in mitochondria and is a key regulatory enzyme in the oxidation of glucose to acetyl-CoA. Phosphorylation of PDC by the pyruvate dehydrogenase kinases (PDK) inhibits its activity. The expression of the pyruvate dehydrogenase kinase 4 (PDK4) gene is increased in fasting and other conditions associated with the switch from the utilization of glucose to fatty acids as an energy source. Transcription of the PDK4 gene is elevated by glucocorticoids and inhibited by insulin. In this study, we have investigated the factors involved in the regulation of the PDK4 gene by these hormones. Glucocorticoids stimulate PDK4 through two glucocorticoid receptor (GR) binding sites located more than 6000 base pairs upstream of the transcriptional start site. Insulin inhibits the glucocorticoid induction in part by causing dissociation of the GR from the promoter. Previously, we found that the estrogen related receptor alpha (ERRalpha) stimulates the expression of PDK4. Here, we determined that one of the ERRalpha binding sites contributes to the insulin inhibition of PDK4. A binding site for the forkhead transcription factor (FoxO1) is adjacent to the ERRalpha binding sites. FoxO1 participates in the glucocorticoid induction of PDK4 and the regulation of this gene by insulin. Our data demonstrate that glucocorticoids and insulin each modulate PDK4 gene expression through complex hormone response units that contain multiple factors.

  10. Na+/K+-ATPase alpha isoforms expression in stroke-prone spontaneously hypertensive rat heart ventricles: effect of salt loading and lacidipine treatment.

    PubMed

    Quintas, Luis Eduardo M; Noël, François; Wibo, Maurice

    2007-06-22

    Changes in myocardial expression of Na+/K+-ATPase alpha-subunit isoforms have been demonstrated in different models of cardiac hypertrophy and hypertension. Here we studied the expression of these isozymes in stroke-prone spontaneously hypertensive rats (SHRSP) and the influence of high salt diet and treatment with the dihydropyridine lacidipine. Adult SHRSP were offered either 1% NaCl or water as drinking solution for 6 weeks. Salt-loaded SHRSP were treated or not with 1 mg/kg/day lacidipine. Compared to Wistar Kyoto (WKY) rats, non-salt-loaded SHRSP presented significant hypertension and cardiac hypertrophy. Salt intake markedly enhanced cardiac hypertrophy, an effect blunted by lacidipine. [3H]Ouabain binding assays on total particulate fractions from heart ventricles revealed the existence of two high-affinity sites with Kd approximately 25 and approximately 200 nM, ascribed to the alpha3 and alpha2 isoforms, respectively. Bmax of alpha3 was unexpectedly high (40% of total high-affinity binding) in ventricles from WKY rats but very low in all groups of SHRSP. On the other hand, Bmax of alpha2 was similar in WKY and non-salt-loaded SHRSP; however, salt loading of SHRSP resulted in a Bmax reduction of 20% (P<0.05), an effect blocked by lacidipine. These effects were largely confirmed by immunoblotting analysis, which, in addition, demonstrated that the density of the ubiquitous alpha1 isoform was comparable among the experimental groups. In conclusion, WKY rats showed a high myocardial expression of the Na+/K+-ATPase alpha3 subunit, which was not found in SHRSP; the level of the alpha2 isoform was similar in untreated SHRSP and WKY; salt-loading of SHRSP promoted reduction of the alpha2 isoform, and this effect was completely hampered by lacidipine.

  11. Evaluation of data discretization methods to derive platform independent isoform expression signatures for multi-class tumor subtyping

    PubMed Central

    2015-01-01

    Background Many supervised learning algorithms have been applied in deriving gene signatures for patient stratification from gene expression data. However, transferring the multi-gene signatures from one analytical platform to another without loss of classification accuracy is a major challenge. Here, we compared three unsupervised data discretization methods--Equal-width binning, Equal-frequency binning, and k-means clustering--in accurately classifying the four known subtypes of glioblastoma multiforme (GBM) when the classification algorithms were trained on the isoform-level gene expression profiles from exon-array platform and tested on the corresponding profiles from RNA-seq data. Results We applied an integrated machine learning framework that involves three sequential steps; feature selection, data discretization, and classification. For models trained and tested on exon-array data, the addition of data discretization step led to robust and accurate predictive models with fewer number of variables in the final models. For models trained on exon-array data and tested on RNA-seq data, the addition of data discretization step dramatically improved the classification accuracies with Equal-frequency binning showing the highest improvement with more than 90% accuracies for all the models with features chosen by Random Forest based feature selection. Overall, SVM classifier coupled with Equal-frequency binning achieved the best accuracy (> 95%). Without data discretization, however, only 73.6% accuracy was achieved at most. Conclusions The classification algorithms, trained and tested on data from the same platform, yielded similar accuracies in predicting the four GBM subgroups. However, when dealing with cross-platform data, from exon-array to RNA-seq, the classifiers yielded stable models with highest classification accuracies on data transformed by Equal frequency binning. The approach presented here is generally applicable to other cancer types for

  12. Characterization of the human SLC2A11 (GLUT11) gene: alternative promoter usage, function, expression, and subcellular distribution of three isoforms, and lack of mouse orthologue.

    PubMed

    Scheepers, Andrea; Schmidt, Stefan; Manolescu, Andrei; Cheeseman, Chris I; Bell, Andreas; Zahn, Claudia; Joost, Hans-Georg; Schürmann, Annette

    2005-01-01

    GLUT11 (SLC2A11) is a class II sugar transport facilitator which exhibits highest similarity with the fructose transporter GLUT5 (about 42%). Here we demonstrate that separate exons 1 (exon 1A, exon 1B, and exon 1C) of the SLC2A11 gene generate mRNAs of three GLUT11 variants (GLUT11-A, GLUT11-B, and GLUT11-C) that differ in the amino acid sequence of their N-termini. All three 5'-flanking regions of exon 1A, exon 1B and exon 1C exhibited promoter activity when expressed as luciferase fusion constructs in COS-7 cells. 5'-RACE-PCR, quantitative real-time PCR, and Northern blot analysis performed with specific probes for exon 1A, 1B and 1C demonstrated that GLUT11-A is expressed in heart, skeletal muscle, and kidney, GLUT11-B in kidney, adipose tissue, and placenta, and GLUT11-C in adipose tissue, heart, skeletal muscle, and pancreas. Surprisingly, mice and rats lack the SLC2A11 gene. When expressed in Xenopus oocytes, all three GLUT11 isoforms transport glucose and fructose but not galactose. There was no apparent difference in the subcellular distribution of the three isoforms expressed in COS-7 cells. Our data indicate that different promoters and splicing of the human SLC2A11 gene generate three GLUT11 isoforms which are expressed in a tissue specific manner but do not appear to differ in their functional characteristics.

  13. Altered Expression Pattern of Acid-Sensing Ion Channel Isoforms in Piriform Cortex After Seizures.

    PubMed

    Wu, Hao; Wang, Chao; Liu, Bei; Li, Huanfa; Zhang, Yu; Dong, Shan; Gao, Guodong; Zhang, Hua

    2016-04-01

    The piriform cortex (PC) is highly susceptible to chemical and electrical seizure induction. Epileptiform activity is associated with an acid shift in extracellular pH, suggesting that acid-sensing ion channels (ASICs) expressed by PC neurons may contribute to this enhanced epileptogenic potential. In epileptic rats and surgical samples from patients with medial temporal lobe epilepsy (TLE), PC layer II ASIC1a-immunopositive neurons appeared swollen with dendritic elongation, and there was loss of ASIC1a-positive neurons in layer III, consistent with enhanced vulnerability to TLE-induced plasticity and cell death. In rats, pilocarpine-induced seizures led to transient downregulation of ASIC1a and concomitant upregulation of ASIC2a in the first few days post-seizure. These changes in expression may be due to seizure-induced oxidative stress as a similar reciprocal change in ASIC1a, and ASIC2a expression was observed in PC12 cells following H2O2 application. The proportion of ASIC1a/ASIC2a heteromers was reduced in the acute phase following status epilepticus (SE) but increased during the latent phase when rats developed spontaneous seizures. Knockdown of ASIC2a by RNAi reduced dendritic length and spine density in primary neurons, suggesting that seizure-induced upregulation of ASIC2a contributes to dendritic lengthening in PC layer II in rats. Administration of the ASIC inhibitor amiloride before pilocarpine reduced the proportion of rats reaching Racine level IV seizures, protected layer II and III neurons, and prolonged survival in the acute phase following SE. Our findings suggest that ASICs may enhance susceptibility to epileptogenesis in the PC. Inhibition of ASICs, particularly ASIC2a, may suppress seizures originating in the PC.

  14. Reduced Peripheral Expression of the Glucocorticoid Receptor α Isoform in Individuals with Posttraumatic Stress Disorder: A Cumulative Effect of Trauma Burden

    PubMed Central

    Morath, Julia; Adenauer, Hannah; Elbert, Thomas; Engler, Harald

    2014-01-01

    Background Posttraumatic stress disorder (PTSD) is a serious psychiatric condition that was found to be associated with altered functioning of the hypothalamic-pituitary-adrenal (HPA) axis and changes in glucocorticoid (GC) responsiveness. The physiological actions of GCs are primarily mediated through GC receptors (GR) of which isoforms with different biological activities exist. This study aimed to investigate whether trauma-experience and/or PTSD are associated with altered expression of GR splice variants. Methods GRα and GRβ mRNA expression levels were determined by real-time quantitative PCR in whole blood samples of individuals with chronic and severe forms of PTSD (n = 42) as well as in ethnically matched reference subjects (non-PTSD, n = 35). Results Individuals suffering from PTSD exhibited significantly lower expression of the predominant and functionally active GRα isoform compared to non-PTSD subjects. This effect remained significant when accounting for gender, smoking, psychotropic medication or comorbid depression. Moreover, the GRα expression level was significantly negatively correlated with the number of traumatic event types experienced, both in the whole sample and within the PTSD patient group. Expression of the less abundant and non-ligand binding GRβ isoform was comparable between patient and reference groups. Conclusions Reduced expression of the functionally active GRα isoform in peripheral blood cells of individuals with PTSD seems to be a cumulative effect of trauma burden rather than a specific feature of PTSD since non-PTSD subjects with high trauma load showed an intermediate phenotype between PTSD patients and individuals with no or few traumatic experiences. PMID:24466032

  15. Expression of nerve growth factor (NGF) isoforms in the rat uterus during pregnancy: accumulation of precursor proNGF.

    PubMed

    Lobos, Edgar; Gebhardt, Claudia; Kluge, Annett; Spanel-Borowski, Katharina

    2005-04-01

    The mechanisms that promote the transient degenerative changes in the uterus innervation during pregnancy remain incompletely understood. Signaling by the nerve growth factor (NGF)-beta is important for maintaining the density of peripheral sympathetic innervation. Here, we analyzed the spatial and temporal expression of NGF isoforms in the rat uterus using RT-PCR, immunoblot analysis, and immunohistochemistry during pregnancy (d 7, 14, and 21), and postpartum (d 1, 8, and 22). Western blot analysis using antibodies to mature NGF-beta and to proNGF domain demonstrated a significant decrease in mature NGF-beta at gestational d 14 and 21 (term pregnancy) and 1 d postpartum, which paralleled a remarkable accumulation of the 26-28-, 32-, and 60-kDa proNGF forms. There were diminished ratios of mature NGF-beta to proNGF independent of uterus growth on the same gestational days. Immunohistochemistry revealed a progressive NGF-beta decline throughout pregnancy in the myometrium and a near absence at term pregnancy, which contrasted with increased NGF immunostaining in the intermyometrial connective tissue layers. More importantly, proNGF-specific antibodies identified the increased NGF immunoreactivity in the intermyometrial layers at term pregnancy as proNGF and not mature NGF-beta. Alterations in the processing of NGF and accumulation of proNGF in the intermyometrial layers, where axonal degeneration occurs, may contribute significantly to the pregnancy-related uterine denervation and to the control of myometrial activity.

  16. C/EBP Transcription Factors in Human Squamous Cell Carcinoma: Selective Changes in Expression of Isoforms Correlate with the Neoplastic State

    PubMed Central

    Anand, Sanjay; Ebner, John; Warren, Christine B.; Raam, Manu S.; Piliang, Melissa; Billings, Steven D.; Maytin, Edward V.

    2014-01-01

    The CCAAT/Enhancer Binding Proteins (C/EBPs) are a family of leucine-zipper transcription factors that regulate physiological processes such as energy metabolism, inflammation, cell cycle, and the development and differentiation of several tissues including skin. Recently, a role for C/EBPs in tumor cell proliferation and differentiation has been proposed, but the incomplete characterization in the literature of multiple translational isoforms of these proteins has made interpretation of these roles difficult. Therefore, we have carefully reexamined C/EBP isoform expression in human non-melanoma skin cancers. C/EBPα, C/EBPβ, and C/EBPδ were analyzed histologically in squamous cell carcinomas (SCC). The individual isoforms of C/EBPα and C/EBPβ were examined by immunofluorescent digital imaging, western blotting and DNA binding activity (electrophoretic mobility shift analysis). Expression of all C/EBP family proteins was decreased in SCC tumors. Suppression was greatest for C/EBPα, less for C/EBPβ, and least for C/EBPδ. Western analyses confirmed that C/EBPα p42 and p30 isoforms were decreased. For C/EBPβ, only the abundant full-length isoform (C/EBPβ−1, LAP*, 55 kD) was reduced, whereas the smaller isoforms, C/EBPβ−2 (LAP, 48 kD) and C/EBPβ−3 (LIP, 20 kD), which are predominantly nuclear, were significantly increased in well- and moderately-differentiated SCC (up to 14-fold for C/EBPβ−3). These elevations correlated with increases in PCNA, a marker of proliferation. Although C/EBPβ displayed increased post-translational modifications in SCC, phosphorylation of C/EBPβ−1 (Thr 235) was not altered. C/EBP-specific DNA binding activity in nuclear and whole-cell extracts of cultured cells and tumors was predominantly attributable to C/EBPβ. In summary, two short C/EBPβ isoforms, C/EBPβ−2 and C/EBPβ−3, represent strong candidate markers for epithelial skin malignancy, due to their preferential expression in carcinoma versus normal skin

  17. PrP mRNA and protein expression in brain and PrP(c) in CSF in Creutzfeldt-Jakob disease MM1 and VV2.

    PubMed

    Llorens, Franc; Ansoleaga, Belén; Garcia-Esparcia, Paula; Zafar, Saima; Grau-Rivera, Oriol; López-González, Irene; Blanco, Rosi; Carmona, Margarita; Yagüe, Jordi; Nos, Carlos; Del Río, José Antonio; Gelpí, Ellen; Zerr, Inga; Ferrer, Isidre

    2013-01-01

    Creutzfeldt-Jakob disease (CJD) is a heterogenic neurodegenerative disorder associated with abnormal post-translational processing of cellular prion protein (PrP(c)). CJD displays distinctive clinical and pathological features which correlate with the genotype at the codon 129 (methionine or valine: M or V respectively) in the prion protein gene and with size of the protease-resistant core of the abnormal prion protein PrP(sc) (type 1: 20/21 kDa and type 2: 19 kDa). MM1 and VV2 are the most common sporadic CJD (sCJD) subtypes. PrP mRNA expression levels in the frontal cortex and cerebellum are reduced in sCJD in a form subtype-dependent. Total PrP protein levels and PrP(sc) levels in the frontal cortex and cerebellum accumulate differentially in sCJD MM1 and sCJD VV2 with no relation between PrP(sc) deposition and spongiform degeneration and neuron loss, but with microgliosis, and IL6 and TNF-α response. In the CSF, reduced PrP(c), the only form present in this compartment, occurs in sCJD MM1 and VV2. PrP mRNA expression is also reduced in the frontal cortex in advanced stages of Alzheimer disease, Lewy body disease, progressive supranuclear palsy, and frontotemporal lobe degeneration, but PrP(c) levels in brain varies from one disease to another. Reduced PrP(c) levels in CSF correlate with PrP mRNA expression in brain, which in turn reflects severity of degeneration in sCJD.

  18. PrP mRNA and protein expression in brain and PrPc in CSF in Creutzfeldt-Jakob disease MM1 and VV2

    PubMed Central

    Llorens, Franc; Ansoleaga, Belén; Garcia-Esparcia, Paula; Zafar, Saima; Grau-Rivera, Oriol; López-González, Irene; Blanco, Rosi; Carmona, Margarita; Yagüe, Jordi; Nos, Carlos; del Río, José Antonio; Gelpí, Ellen; Zerr, Inga; Ferrer, Isidre

    2013-01-01

    Creutzfeldt-Jakob disease (CJD) is a heterogenic neurodegenerative disorder associated with abnormal post-translational processing of cellular prion protein (PrPc). CJD displays distinctive clinical and pathological features which correlate with the genotype at the codon 129 (methionine or valine: M or V respectively) in the prion protein gene and with size of the protease-resistant core of the abnormal prion protein PrPsc (type 1: 20/21 kDa and type 2: 19 kDa). MM1 and VV2 are the most common sporadic CJD (sCJD) subtypes. PrP mRNA expression levels in the frontal cortex and cerebellum are reduced in sCJD in a form subtype-dependent. Total PrP protein levels and PrPsc levels in the frontal cortex and cerebellum accumulate differentially in sCJD MM1 and sCJD VV2 with no relation between PrPsc deposition and spongiform degeneration and neuron loss, but with microgliosis, and IL6 and TNF-α response. In the CSF, reduced PrPc, the only form present in this compartment, occurs in sCJD MM1 and VV2. PrP mRNA expression is also reduced in the frontal cortex in advanced stages of Alzheimer disease, Lewy body disease, progressive supranuclear palsy, and frontotemporal lobe degeneration, but PrPc levels in brain varies from one disease to another. Reduced PrPc levels in CSF correlate with PrP mRNA expression in brain, which in turn reflects severity of degeneration in sCJD. PMID:24047819

  19. Expression of 11β-hydroxysteroid dehydrogenase isoforms in canine adrenal glands treated with trilostane.

    PubMed

    Teshima, Takahiro; Matsumoto, Hirotaka; Kumagai, Takayuki; Kurano, Mai; Koyama, Hidekazu

    2014-06-01

    Trilostane, a competitive inhibitor of 3β-hydroxysteroid dehydrogenase, is often used to treat canine hyperadrenocorticism. In some species, trilostane has been shown to have additional effects on steroid biosynthesis, and it has been postulated that trilostane might have effects on 11β-hydroxysteroid dehydrogenase (11β-HSD) in dogs. To investigate the effect of trilostane on 11β-HSD in canine adrenal glands, healthy Beagle dogs were treated with trilostane for 8 weeks. Trilostane treatment resulted in a significant decrease of the cortisol/cortisone ratio in the serum. The adrenal gland mRNA and protein expression levels of 11β-HSD type 1 and 11β-HSD type 2 were significantly higher and significantly lower respectively in dogs treated with trilostane compared to those in control healthy Beagle dogs. These findings suggest that trilostane may have an effect on 11β-HSD activity in canine adrenal glands.

  20. Isoform-level brain expression profiling of the spermidine/spermine N1-Acetyltransferase1 (SAT1) gene in major depression and suicide.

    PubMed

    Pantazatos, Spiro P; Andrews, Stuart J; Dunning-Broadbent, Jane; Pang, Jiuhong; Huang, Yung-Yu; Arango, Victoria; Nagy, Peter L; John Mann, J

    2015-07-01

    Low brain expression of the spermidine/spermine N-1 acetyltransferase (SAT1) gene, the rate-limiting enzyme involved in catabolism of polyamines that mediate the polyamine stress response (PSR), has been reported in depressed suicides. However, it is unknown whether this effect is associated with depression or with suicide and whether all or only specific isoforms expressed by SAT1, such as the primary 171 amino acid protein-encoding transcript (SSAT), or an alternative splice variant (SSATX) that is involved in SAT1 regulated unproductive splicing and transcription (RUST), are involved. We applied next generation sequencing (RNA-seq) to assess gene-level, isoform-level, and exon-level SAT1 expression differences between healthy controls (HC, N = 29), DSM-IV major depressive disorder suicides (MDD-S, N = 21) and MDD non-suicides (MDD, N = 9) in the dorsal lateral prefrontal cortex (Brodmann Area 9, BA9) of medication-free individuals postmortem. Using small RNA-seq, we also examined miRNA species putatively involved in SAT1 post-transcriptional regulation. A DSM-IV diagnosis was made by structured interview. Toxicology and history ruled out recent psychotropic medication. At the gene-level, we found low SAT1 expression in both MDD-S (vs. HC, p = 0.002) and MDD (vs. HC, p = 0.002). At the isoform-level, reductions in MDD-S (vs. HC) were most pronounced in four transcripts including SSAT and SSATX, while reductions in MDD (vs. HC) were pronounced in three transcripts, one of which was reduced in MDD relative to MDD-S (all p < 0.1 FDR corrected). We did not observe evidence for differential exon-usage (i.e. splicing) nor differences in miRNA expression. Results replicate the finding of low SAT1 brain expression in depressed suicides in an independent sample and implicate low SAT1 brain expression in MDD independent of suicide. Low expressions of both SSAT and SATX isoforms suggest that shared transcriptional mechanisms involved in RUST may account for low SAT1 brain

  1. Species differences in cannabinoid receptor 2 (CNR2 gene): identification of novel human and rodent CB2 isoforms, differential tissue expression, and regulation by cannabinoid receptor ligands

    PubMed Central

    Liu, Qing-Rong; Pan, Chun-Hung; Hishimoto, Akitoyo; Li, Chuan-Yun; Xi, Zheng-Xiong; Llorente-Berzal, Alvaro; Viveros, Maria-Paz; Ishiguro, Hiroki; Arinami, Tadao; Onaivi, Emmanuel Shan; Uhl, George R.

    2009-01-01

    Cannabinoids, endocannabinoids and marijuana activate two well-characterized cannabinoid receptors (CBRs), CB1-Rs and CB2-Rs. The expression of CB1-Rs in the brain and periphery has been well studied but neuronal CB2-Rs have received much less attention than CB1-Rs. Many studies have now identified and characterized functional glial and neuronal CB2-Rs in the central nervous system. However, many features of CB2-R gene structure, regulation and variation remain poorly characterized in comparison to the CB1-R. Here, we report on the discovery of a novel human CB2 gene promoter encoding testis (CB2A) isoform with starting exon located ca 45 kb upstream from the previously identified promoter encoding the spleen isoform (CB2B). The 5′ exons of both CB2 isoforms are untranslated 5′UTRs and alternatively spliced to the major protein coding exon of the CB2 gene. CB2A is expressed higher in testis and brain than CB2B that is expressed higher in other peripheral tissues than CB2A. Species comparison found that the CB2 gene of human, rat and mouse genomes deviated in their gene structures and isoform expression patterns. mCB2A expression was increased significantly in the cerebellum of mice treated with the CB-R mixed agonist, WIN55212-2. These results provide much improved information about CB2 gene structure and its human and rodent variants that should be considered in developing CB2-R-based therapeutic agents. PMID:19496827

  2. The Crumbs_C isoform of Drosophila shows tissue- and stage-specific expression and prevents light-dependent retinal degeneration

    PubMed Central

    Spannl, Stephanie; Kumichel, Alexandra; Hebbar, Sarita; Kapp, Katja; Gonzalez-Gaitan, Marcos; Winkler, Sylke; Blawid, Rosana; Jessberger, Gregor

    2017-01-01

    ABSTRACT Drosophila Crumbs (Crb) is a key regulator of epithelial polarity and fulfils a plethora of other functions, such as growth regulation, morphogenesis of photoreceptor cells and prevention of retinal degeneration. This raises the question how a single gene regulates such diverse functions, which in mammals are controlled by three different paralogs. Here, we show that in Drosophila different Crb protein isoforms are differentially expressed as a result of alternative splicing. All isoforms are transmembrane proteins that differ by just one EGF-like repeat in their extracellular portion. Unlike Crb_A, which is expressed in most embryonic epithelia from early stages onward, Crb_C is expressed later and only in a subset of embryonic epithelia. Flies specifically lacking Crb_C are homozygous viable and fertile. Strikingly, these flies undergo light-dependent photoreceptor degeneration despite the fact that the other isoforms are expressed and properly localised at the stalk membrane. This allele now provides an ideal possibility to further unravel the molecular mechanisms by which Drosophila crb protects photoreceptor cells from the detrimental consequences of light-induced cell stress. PMID:28202468

  3. Effects of nutrition and genotype on prion protein (PrPC) gene expression in the fetal and maternal sheep placenta.

    PubMed

    Evoniuk, J M; Johnson, M L; Borowicz, P P; Caton, J S; Vonnahme, K A; Reynolds, L P; Taylor, J B; Stoltenow, C L; O'Rourke, K I; Redmer, D A

    2008-05-01

    For placental transmission of scrapie to occur, the normal cellular prion protein (PrPC) must be converted to an abnormal infectious form known as PrPSc. PrPC genotype influences susceptibility to contracting scrapie, but we still do not understand whether genotype or expression levels of PrPC are important in transmission of scrapie. Some evidence exists that nutrition affects expression levels of PrPC. Thus, we evaluated the effects of genotype and nutrition on PrPC mRNA and protein expression in adolescent ewes fed at control (100% of National Research Council [NRC] requirements) or restricted (60% of NRC) levels of diet intake during two periods of pregnancy (days 50-90 and days 90-130)]. Gravid uteri (n=50) from singleton pregnancies were collected at day 130, and placentomes were either separated into caruncular (CAR; maternal) or cotyledonary (COT; fetal) placenta and snap-frozen for PrPC mRNA expression or perfusion fixed for PrPC protein expression. PrPC genotypes were determined (codons 136 and 171) using SNP assay. There were no genotype effects on PrPC mRNA expression in CAR or on PrPC protein expression in either CAR or COT, but PrPC mRNA expression in COT was greater (P<0.02) when codon 136 was homozygous for alanine. Some PrPC protein-positive cells were found in the epithelium of CAR, but most were found in trophoblast binucleate and mononucleate cells of COT. In CAR, from days 90 to 130, PrPC protein abundance was greater (P=0.003) in diet-restricted ewes than in control ewes, but was less uniformly distributed (P<0.007). Additionally, in COT, from days 90 to 130, PrPC protein was less uniformly distributed (P<0.01) in diet-restricted ewes. The localized increase in PrPC protein expression, found in ewes diet-restricted late in pregnancy, may suggest a protective role for PrPC in placental biology. Further study is needed to evaluate whether nutrition, PrPC genotype, and PrPC expression levels influence placental transmission of scrapie.

  4. Creb1-Mecp2-mCpG Complex Transactivates Postnatal Murine Neuronal Glucose Transporter Isoform 3 Expression

    PubMed Central

    Chen, Yongjun; Shin, Bo-Chul; Thamotharan, Shanthie

    2013-01-01

    The murine neuronal facilitative glucose transporter isoform 3 (Glut3) is developmentally regulated, peaking in expression at postnatal day (PN)14. In the present study, we characterized a canonical CpG island spanning the 5′-flanking region of the glut3 gene. Methylation-specific PCR and bisulfite sequencing identified methylation of this CpG (mCpG) island of the glut3 gene, frequency of methylation increasing 2.5-fold with a 1.6-fold increase in DNA methyl transferase 3a concentrations noted with advancing postnatal age (PN14 vs PN3). 5′-flanking region of glut3-luciferase reporter transient transfection in HT22 hippocampal neurons demonstrated that mCpGs inhibit glut3 transcription. Contrary to this biological function, glut3 expression rises synchronously with mCpGs in PN14 vs PN3 neurons. Chromatin immunoprecipitation (IP) revealed that methyl-CpG binding protein 2 (Mecp2) bound the glut3-mCpGs. Depending on association with specific coregulators, Mecp2, a dual regulator of gene transcription, may repress or activate a downstream gene. Sequential chromatin IP uncovered the glut3-mCpGs to bind Mecp2 exponentially upon recruitment of Creb1 rather than histone deacetylase 1. Co-IP and coimmunolocalization confirmed that Creb1 associated with Mecp2 and cotransfection with glut3-mCpG in HT22 cells enhanced glut3 transcription. Separate 5-aza-2′-deoxycytidine pretreatment or in combination with trichostatin A reduced mCpG and specific small interference RNAs targeting Mecp2 and Creb1 separately or together depleting Mecp2 and/or Creb1 binding of glut3-mCpGs reduced glut3 expression in HT22 cells. We conclude that Glut3 is a methylation-sensitive neuronal gene that recruits Mecp2. Recruitment of Creb1-Mecp2 by glut3-mCpG contributes towards transactivation, formulating an escape from mCpG-induced gene suppression, and thereby promoting developmental neuronal glut3 gene transcription and expression. PMID:23493374

  5. Distribution of estrogen and progesterone receptors isoforms in endometrial cancer

    PubMed Central

    2014-01-01

    Background 70–80% of sporadic endometrial carcinomas are defined as endometrioid carcinoma (EC). Early-stage, well differentiated endometrial carcinomas usually retain expression of estrogen and progesterone receptors (ER and PR, respectively), as advanced stage, poorly differentiated tumors often lack one or both of these receptors. Well-described EC prognosis includes tumor characteristics, such as depth of myometrial invasion. Therefore, in the current study, we evaluated the expression profile of ER and PR isoforms, including ER-α, PR-A and PR–B, in correlation to EC tumor histological depth. Methods Using immunohistochemistry and image analysis software, the expression of ER-α, PR-A, PR–B and Ki67 was assessed in endometrial stroma and epithelial glands of superficial, deep and extra-tumoral sections of 15 paraffin embedded EC specimens, and compared to 5 biopsies of non-malignant endometrium. Results Expression of PR-A and ER-α was found to be lower in EC compared to nonmalignant tissue, as the stromal expression was dramatically reduced compared to epithelial cells. Expression ratios of both receptors were significantly high in superficial and deep portions of EC; in non-tumoral portion of EC were close to the ratios of nonmalignant endometrium. PR-B expression was low in epithelial glands of EC superficial and deep portions, and high in the extra-tumoral region. Elevated PR-B expression was found in stroma of EC, as well. Conclusions The ratio of ER-α and PR-A expression in the epithelial glands and the stroma of EC biopsies may serve as an additional parameter in the histological evaluation of EC tumor. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1155060506119016 PMID:24684970

  6. SVCT2 transporter expression is post-natally induced in cortical neurons and its function is regulated by its short isoform.

    PubMed

    Salazar, Katterine; Cerda, Gustavo; Martínez, Fernando; Sarmiento, José M; González, Carlos; Rodríguez, Federico; García-Robles, María; Tapia, Juan Carlos; Cifuentes, Manuel; Nualart, Francisco

    2014-09-01

    Different studies have demonstrated the importance of micronutrients, such as vitamins, for normal adult brain function and development. Vitamin C is not synthesized in the brain, but high levels are detected in this organ because of the existence of specific uptake mechanisms, which concentrate ascorbic acid from the bloodstream to the cerebrospinal fluid and then into neurons and glial cells. Two different isoforms of sodium-vitamin C cotransporters (SVCT1 and SVCT2) have been cloned. SVCT2 expression has been observed in the adult hippocampus and cortical neurons by in situ hybridization. In addition, the localization of SVCT2 in the rat fetal brain has been studied by immunohistochemistry and in situ hybridization, demonstrating that SVCT2 is highly expressed in the ventricular and subventricular areas of the brain cortex. However, there are currently no immunohistochemical data regarding SVCT2 expression and function in the post-natal brain. Therefore, we analyzed SVCT2 expression in the developing brain cortex of mice, and demonstrated an increase in SVCT2 mRNA in mice at 1-15 days of age. The expression of a short isoform, SVCT2sh, was also detected within the same period. SVCT2 expression was concentrated in neurons within the inner layer of the brain cortex. Both SVCT2 isoforms were coexpressed in N2a cells to obtain functional data. Fluorescence resonance energy transfer analysis revealed a molecular interaction between SVCT2wt and SVCT2sh. Finally, differences in transport ratios suggested that SVCT2sh expression inhibited ascorbic acid uptake in N2a cells when both isoforms were coexpressed. The sodium-vitamin C cotransporter, SVCT2, is induced in neurons within the inner layer of the brain cortex during post-natal development, mainly in pyramidal cortex neurons. Two different isoforms, SVCT2wt and SVCT2sh, were detected. Using in vitro studies, we suggest a molecular interaction between SVCT2wt and SVCT2sh, which may regulate the affinity of vitamin C

  7. Expression of the TPα and TPβ isoforms of the thromboxane prostanoid receptor (TP) in prostate cancer: clinical significance and diagnostic potential

    PubMed Central

    Mulvaney, Eamon P.; Shilling, Christine; Eivers, Sarah B.; Perry, Antoinette S.; Bjartell, Anders; Kay, Elaine W.; Watson, R. William; Kinsella, B. Therese

    2016-01-01

    The prostanoid thromboxane (TX) A2 plays a central role in haemostasis and is increasingly implicated in cancer progression. TXA2 signals through two T Prostanoid receptor (TP) isoforms termed TPα and TPβ, with both encoded by the TBXA2R gene. Despite exhibiting several functional and regulatory differences, the role of the individual TP isoforms in neoplastic diseases is largely unknown. This study evaluated expression of the TPα and TPβ isoforms in tumour microarrays of the benign prostate and different pathological (Gleason) grades of prostate cancer (PCa). Expression of TPβ was significantly increased in PCa relative to benign tissue and strongly correlated with increasing Gleason grade. Furthermore, higher TPβ expression was associated with increased risk of biochemical recurrence (BCR) and significantly shorter disease-free survival time in patients post-surgery. While TPα was more variably expressed than TPβ in PCa, increased/high TPα expression within the tumour also trended toward increased BCR and shorter disease-free survival time. Comparative genomic CpG DNA methylation analysis revealed substantial differences in the extent of methylation of the promoter regions of the TBXA2R that specifically regulate expression of TPα and TPβ, respectively, both in benign prostate and in clinically-derived tissue representative of precursor lesions and progressive stages of PCa. Collectively, TPα and TPβ expression is differentially regulated both in the benign and tumourigenic prostate, and coincides with clinical pathology and altered CpG methylation of the TBXA2R gene. Analysis of TPβ, or a combination of TPα/TPβ, expression levels may have significant clinical potential as a diagnostic biomarker and predictor of PCa disease recurrence. PMID:27689401

  8. Changes in Expression of Connexin Isoforms in the Caudal Epididymis of Adult Sprague-Dawley Rats exposed to Estradiol Benzoate or Flutamide at the Neonatal Age

    PubMed Central

    Lee, Ki-Ho

    2016-01-01

    Direct communication between neighboring cells via gap junction in tissue is important for maintenance and regulation of its physiological functions. Each epididymal region has different composition of cell types. It is well recognized that the epididymis is a steroid hormone-responsive tissue. The present study was designed to determine the effect of estradiol benzoate (EB) or flutamide exposured at the early postnatal age on the expression of connexin (Cx) isoforms in the caudal epididymis. The EB or flutamide was subcutaneously administrated to male Spragure Dawley rat at 7 days of age, and expressional changes of Cx isoforms in the adult corpus epididymis were determined by quantitative real-time PCR. The treatment of low-dose EB resulted in decreases of Cx30.3, Cx31.1, Cx37, and Cx45 expression but caused an increase of Cx32 expression. Exposure to high-dose EB led into expressional increases of Cx31, Cx31.1, Cx32, Cx40, and Cx43, even though a decrease of Cx37 expression was found with a high-dose EB treatment. A low-dose flutamide induced increases of Cx31, Cx31.1, Cx32, and Cx43 expression but a decrease of Cx37 expression. Expression of most Cx genes were significantly increased by a high-dose flutamide, while no expressional change of Cx26 and Cx40 was detected by a high-dose flutamide. These results indicate that expression of Cx isoforms in the caudal epididymis is altered by exposure to steroidal compounds at the prepubertal age. It is suggested that a contact with environmental exogenous materials during the early postnatal period would lead to alteration of epididymal functions at the adult. PMID:27796005

  9. Expression of the Minor Isoform Pea Ferredoxin in Tobacco Alters Photosynthetic Electron Partitioning and Enhances Cyclic Electron Flow1[W

    PubMed Central

    Blanco, Nicolás E.; Ceccoli, Romina D.; Vía, María V. Dalla; Voss, Ingo; Segretin, María E.; Bravo-Almonacid, Fernando F.; Melzer, Michael; Hajirezaei, Mohammad-Reza; Scheibe, Renate; Hanke, Guy T.

    2013-01-01

    Ferredoxins (Fds) are ferrosulfoproteins that function as low-potential electron carriers in plants. The Fd family is composed of several isoforms that share high sequence homology but differ in functional characteristics. In leaves, at least two isoforms conduct linear and cyclic photosynthetic electron transport around photosystem I, and mounting evidence suggests the existence of at least partial division of duties between these isoforms. To evaluate the contribution of different kinds of Fds to the control of electron fluxes along the photosynthetic electron transport chain, we overexpressed a minor pea (Pisum sativum) Fd isoform (PsFd1) in tobacco (Nicotiana tabacum) plants. The transplastomic OeFd1 plants exhibited variegated leaves and retarded growth and developmental rates. Photosynthetic studies of these plants indicated a reduction in carbon dioxide assimilation rates, photosystem II photochemistry, and linear electron flow. However, the plants showed an increase in nonphotochemical quenching, better control of excitation pressure at photosystem II, and no evidence of photoinhibition, implying a better dynamic regulation to remove excess energy from the photosynthetic electron transport chain. Finally, analysis of P700 redox status during illumination confirmed that the minor pea Fd isoform promotes enhanced cyclic flow around photosystem I. The two novel features of this work are: (1) that Fd levels achieved in transplastomic plants promote an alternative electron partitioning even under greenhouse light growth conditions, a situation that is exacerbated at higher light intensity measurements; and (2) that an alternative, minor Fd isoform has been overexpressed in plants, giving new evidence of labor division among Fd isoforms. PMID:23370717

  10. Major isoform of zebrafish P0 is a 23.5 kDa myelin glycoprotein expressed in selected white matter tracts of the central nervous system.

    PubMed

    Bai, Qing; Sun, Ming; Stolz, Donna B; Burton, Edward A

    2011-06-01

    The zebrafish mpz gene, encoding the ortholog of mammalian myelin protein zero, is expressed in oligodendrocytes of the zebrafish central nervous system (CNS). The putative gene product, P0, has been implicated in promoting axonal regeneration in addition to its proposed structural functions in compact myelin. We raised novel zebrafish P0-specific antibodies and established that P0 is a 23.5 kDa glycoprotein containing a 3 kDa N-linked carbohydrate moiety. P0 was localized to myelin sheaths surrounding axons, but was not detected in the cell bodies or proximal processes of oligodendrocytes. Many white matter tracts in the adult zebrafish CNS were robustly immunoreactive for P0, including afferent visual and olfactory pathways, commissural and longitudinal tracts of the brain, and selected ascending and descending tracts of the spinal cord. P0 was first detected during development in premyelinating oligodendrocytes of the ventral hindbrain at 48 hours postfertilization (hpf). By 72 hpf, short segments of longitudinally oriented P0-immunoreactive myelinating axons were seen in the hindbrain; expression in the spinal cord, optic pathways, hindbrain commissures, midbrain, and peripheral nervous system followed. The mpz transcript was found to be alternatively spliced, giving rise to P0 isoforms with alternative C-termini. The 23.5 kDa isoform was most abundant in the CNS, but other isoforms predominated in the myelin sheath surrounding the Mauthner axon. These data provide a detailed account of P0 expression and demonstrate novel P0 isoforms, which may have discrete functional properties. The restriction of P0 immunoreactivity to myelin sheaths indicates that the protein is subject to stringent intracellular compartmentalization, which likely occurs through posttranslational mechanisms.

  11. Isoform-specific regulation of transforming growth factor-β mRNA expression in macrophages in response to adrenoceptor stimulation.

    PubMed

    Yanagawa, Yoshiki; Hiraide, Sachiko; Iizuka, Kenji

    2016-01-01

    Transforming growth factor-beta (TGF-β) is a multifunctional cytokine responsible for both immune regulation and tissue repair. Although TGF-β consists of TGF-β1, -β2, and -β3 in mammals, isoform-selective transcriptional regulation is less well documented in myeloid linage cells such as macrophages. In the present study, the effect of the stress-related catecholamine adrenaline on the expression of TGF-β isoforms in RAW264.7 macrophages and murine bone marrow-derived macrophages was examined. Treatment with adrenaline markedly increased the mRNA expression of TGF-β3 but not of TGF-β1 and -β2. Agonist and antagonist studies indicated that adrenaline-induced TGF-β3 mRNA expression is mediated via β2 -adrenoceptor. Protein kinase A (PKA) inhibitor H89 was found to block an increase in adrenoceptor-mediated TGF-β3 mRNA expression. The membrane-permeable cAMP analog 8-Br-cAMP increased the mRNA expression of TGF-β3 but not of TGF-β1 and -β2. Thus, the β2 -adrenoceptor-mediated cAMP-PKA pathway appears to enhance TGF-β3 mRNA expression in macrophages. Adrenoceptor-mediated TGF-β3 expression by macrophages may influence immune regulation and tissue repair in conditions of stress, during which the sympathetic-nervous system releases catecholamines.

  12. NFAT2 Isoforms Differentially Regulate Gene Expression, Cell Death, and Transformation through Alternative N-Terminal Domains

    PubMed Central

    Lucena, Pedro I.; Faget, Douglas V.; Pachulec, Emilia; Robaina, Marcela C.; Klumb, Claudete E.

    2015-01-01

    The NFAT (nuclear factor of activated T cells) family of transcription factors is composed of four calcium-responsive proteins (NFAT1 to -4). The NFAT2 (also called NFATc1) gene encodes the isoforms NFAT2α and NFAT2β that result mainly from alternative initiation exons that provide two different N-terminal transactivation domains. However, the specific roles of the NFAT2 isoforms in cell physiology remain unclear. Because previous studies have shown oncogenic potential for NFAT2, this study emphasized the role of the NFAT2 isoforms in cell transformation. Here, we show that a constitutively active form of NFAT2α (CA-NFAT2α) and CA-NFAT2β distinctly control death and transformation in NIH 3T3 cells. While CA-NFAT2α strongly induces cell transformation, CA-NFAT2β leads to reduced cell proliferation and intense cell death through the upregulation of tumor necrosis factor alpha (TNF-α). CA-NFAT2β also increases cell death and upregulates Fas ligand (FasL) and TNF-α in CD4+ T cells. Furthermore, we demonstrate that differential roles of NFAT2 isoforms in NIH 3T3 cells depend on the N-terminal domain, where the NFAT2β-specific N-terminal acidic motif is necessary to induce cell death. Interestingly, the NFAT2α isoform is upregulated in Burkitt lymphomas, suggesting an isoform-specific involvement of NFAT2 in cancer development. Finally, our data suggest that alternative N-terminal domains of NFAT2 could provide differential mechanisms for the control of cellular functions. PMID:26483414

  13. NFAT2 Isoforms Differentially Regulate Gene Expression, Cell Death, and Transformation through Alternative N-Terminal Domains.

    PubMed

    Lucena, Pedro I; Faget, Douglas V; Pachulec, Emilia; Robaina, Marcela C; Klumb, Claudete E; Robbs, Bruno K; Viola, João P B

    2016-01-01

    The NFAT (nuclear factor of activated T cells) family of transcription factors is composed of four calcium-responsive proteins (NFAT1 to -4). The NFAT2 (also called NFATc1) gene encodes the isoforms NFAT2α and NFAT2β that result mainly from alternative initiation exons that provide two different N-terminal transactivation domains. However, the specific roles of the NFAT2 isoforms in cell physiology remain unclear. Because previous studies have shown oncogenic potential for NFAT2, this study emphasized the role of the NFAT2 isoforms in cell transformation. Here, we show that a constitutively active form of NFAT2α (CA-NFAT2α) and CA-NFAT2β distinctly control death and transformation in NIH 3T3 cells. While CA-NFAT2α strongly induces cell transformation, CA-NFAT2β leads to reduced cell proliferation and intense cell death through the upregulation of tumor necrosis factor alpha (TNF-α). CA-NFAT2β also increases cell death and upregulates Fas ligand (FasL) and TNF-α in CD4(+) T cells. Furthermore, we demonstrate that differential roles of NFAT2 isoforms in NIH 3T3 cells depend on the N-terminal domain, where the NFAT2β-specific N-terminal acidic motif is necessary to induce cell death. Interestingly, the NFAT2α isoform is upregulated in Burkitt lymphomas, suggesting an isoform-specific involvement of NFAT2 in cancer development. Finally, our data suggest that alternative N-terminal domains of NFAT2 could provide differential mechanisms for the control of cellular functions.

  14. Uncoupling PR gene expression from NPR1 and bacterial resistance: characterization of the dominant Arabidopsis cpr6-1 mutant.

    PubMed Central

    Clarke, J D; Liu, Y; Klessig, D F; Dong, X

    1998-01-01

    In Arabidopsis, NPR1 mediates the salicylic acid (SA)-induced expression of pathogenesis-related (PR) genes and systemic acquired resistance (SAR). Here, we report the identification of another component, CPR 6, that may function with NPR1 in regulating PR gene expression. The dominant CPR 6-1 mutant expresses the SA/NPR1-regulated PR genes (PR-1, BGL 2, and PR-5) and displays enhanced resistance to Pseudomonas syringae pv maculicola ES4326 and Peronospora parasitica Noco2 in the absence of SAR induction. cpr 6-1-induced PR gene expression is not suppressed in the cpr 6-1 npr1-1 double mutant but is suppressed when SA is removed by salicylate hydroxylase. Thus, constitutive PR gene expression in cpr 6-1 requires SA but not NPR1. In addition, resistance to P. s. maculicola ES4326 is suppressed in the cpr 6-1 npr1-1 double mutant, despite expression of PR-1, BGL 2, and PR-5. Resistance to P. s. maculicola ES4326 must therefore be accomplished through unidentified antibacterial gene products that are regulated through NPR1. These results show that CPR 6 is an important regulator of multiple signal transduction pathways involved in plant defense. PMID:9548982

  15. Tissue-specific alternative promoters regulate the expression of the two sarco/endoplasmic reticulum Ca-ATPase isoforms from Artemia franciscana.

    PubMed

    Escalante, R; Sastre, L

    1995-10-01

    The sarco/endoplasmic reticulum Ca-ATPase gene from Artemia franciscana is transcribed into two mRNAs of 4.5 and 5.2 kb that code for protein isoforms differing at their carboxyl terminus. Northern blot assays and anchored polymerase chain reaction (PCR) experiments have shown that these two mRNAs also differ at the initial part of their 5' untranslated region. The 5.2-kb mRNA-specific 5' untranslated region is present as an independent exon whose transcription is regulated by a promoter different from the one previously described that regulates the expression of the 4.5-kb mRNA. The nucleotide sequence of the 5.2-kb mRNA promoter and the transcription initiation site have been determined. These results suggest that the expression of the two protein isoforms is regulated in A. franciscana at the transcription initiation step, in contrast with the vertebrates sarco/endoplasmic reticulum Ca-ATPase genes 1 and 2 which have unique promoters for transcription of the two isoforms encoded by each gene.

  16. A novel alternative spliced Mpv17-like protein isoform localizes in cytosol and is expressed in a kidney- and adult-specific manner.

    PubMed

    Iida, Reiko; Yasuda, Toshihiro; Tsubota, Etsuko; Takatsuka, Hisakazu; Masuyama, Mika; Matsuki, Takasumi; Kishi, Koichiro

    2005-01-01

    Mpv17-like protein (M-LP) has been identified as a new protein that shows high sequence homology with Mpv17 protein, a peroxisomal membrane protein involved in the development of early onset glomerulosclerosis. We previously showed that the originally identified M-LP isoform, designated M-LPL, is, like Mpv17, localized in peroxisomes, and that transfection with M-LPL up-regulates expression of the manganese superoxide dismutase (SOD2) gene [R. Iida, T. Yasuda, E. Tsubota, H. Takatsuka, M. Masuyama, T. Matsuki, K. Kishi, M-LP, Mpv17-like protein, has a peroxisomal membrane targeting signal comprising a transmembrane domain and a positively charged loop and up-regulates expression of the manganese superoxide dismutase gene. J. Biol. Chem. 278 (2003) 6301-6306.]. We report here the identification of a novel alternative splicing product of the M-LP gene, designated M-LPS. A comparison of the genomic sequence with the cDNA sequences and an analysis of 5'-flanking regions revealed that the two isoforms are generated by alternative usage of two promoters. M-LPS consists of the C-terminal half of M-LPL (90 amino acids) and therefore lacks the peroxisome targeting signal of membrane protein that exists near the N-terminus of M-LPL. Expression of green fluorescent protein-tagged M-LPS in COS-7 cells demonstrated that M-LPS localizes in the cytosol. In mice, M-LPS is expressed exclusively in kidneys after the age of 6 weeks. Moreover, quantitative real-time PCR analysis revealed that transfection with M-LPS up-regulates expression of the SOD2 gene and down-regulates expression of the cellular glutathione peroxidase (Gpx1) and plasma glutathione peroxidase (Gpx3) genes. Taken together, these results suggest different functional attributes of the two M-LP isoforms during aging and development.

  17. Homozygous truncating mutation in prenatally expressed skeletal isoform of TTN gene results in arthrogryposis multiplex congenita and myopathy without cardiac involvement.

    PubMed

    Fernández-Marmiesse, Ana; Carrascosa-Romero, M Carmen; Alfaro Ponce, Blanca; Nascimento, Andres; Ortez, Carlos; Romero, Norma; Palacios, Lourdes; Jimenez-Mallebrera, Cecilia; Jou, Cristina; Gouveia, Sofía; Couce, María L

    2017-02-01

    We report the case of a newborn with arthrogryposis multiplex congenita and severe axial hypotonia without cardiac involvement in which, using a customized targeted next-generation sequencing assay for 64 myopathy-associated genes, we detected a novel homozygous truncating mutation, c.38661_38665del, in exon 197 of the TTN gene that is expressed only in the fetal skeletal isoform. Its pathogenicity is supported by evidence of maternal isodisomy for chromosome 2. Muscle pathology showed fibers with core-like areas devoid of oxidative staining and cytoplasmic bodies. Electron microscopy showed the replacement of the sarcomeric structure with filamentous material. Identification of this mutation expands the phenotypic spectrum of the TTN gene and shows for the first time that a mutation not found in adult TTN isoforms is involved in the development of a neuromuscular disorder. TTN mutations should be considered in all severe congenital myopathies with arthrogryposis without cardiac involvement.

  18. Cloning of a brain-type isoform of human Rab GDI and its expression in human neuroblastoma cell lines and tumor specimens.

    PubMed

    Nishimura, N; Goji, J; Nakamura, H; Orita, S; Takai, Y; Sano, K

    1995-11-15

    Rab proteins, a family of Ras-related small GTP-binding proteins, play a key role in regulating intracellular vesicle trafficking. Rab GDP dissociation inhibitor (GDI3) forms a soluble complex with Rab proteins and thereby prevents the exchange of GDP for GTP. Recently, two isoforms of Rab GDI cDNA were isolated from rats and mice. In this study, we have isolated a brain-type isoform of human Rab GDI cDNA and examined its expression in neuroblastoma. We tentatively designate it as human Rab GDI alpha (hu GDI alpha) and another human Rab GDI, as human Rab GDI beta (hu GDI beta). Hu GDI alpha cDNA encodes a protein of 447 amino acids with a deduced molecular weight of 50,200. Northern blot analysis revealed that hu GDI alpha gene is expressed abundantly in the brain but much less in other tissues, while hu GDI beta gene is ubiquitously expressed. All human neuroblastoma cell lines and tumor specimens examined express hu GDI alpha gene to various extents, while a human T cell leukemia cell line, MOLT3, does not. The levels of both hu GDI alpha and beta mRNA were constant in a human neuroblastoma cell line, NB1, during its neuronal differentiation, while Rab3A and neurofilament-L gene expression and the number of neurosecretory granules were elevated at this condition. These results suggest that hu GDI alpha gene expression is not related to the differentiation state of neuronal cells.

  19. Heterologous expression of a rice metallothionein isoform (OsMTI-1b) in Saccharomyces cerevisiae enhances cadmium, hydrogen peroxide and ethanol tolerance.

    PubMed

    Ansarypour, Zahra; Shahpiri, Azar

    2017-02-04

    Metallothioneins are a superfamily of low-molecular-weight, cysteine (Cys)-rich proteins that are believed to play important roles in protection against metal toxicity and oxidative stress. The main purpose of this study was to investigate the effect of heterologous expression of a rice metallothionein isoform (OsMTI-1b) on the tolerance of Saccharomyces cerevisiae to Cd(2+), H2O2 and ethanol stress. The gene encoding OsMTI-1b was cloned into p426GPD as a yeast expression vector. The new construct was transformed to competent cells of S. cerevisiae. After verification of heterologous expression of OsMTI-1b, the new strain and control were grown under stress conditions. In comparison to control strain, the transformed S. cerevisiae cells expressing OsMTI-1b showed more tolerance to Cd(2+) and accumulated more Cd(2+) ions when they were grown in the medium containing CdCl2. In addition, the heterologous expression of GST-OsMTI-1b conferred H2O2 and ethanol tolerance to S. cerevisiae cells. The results indicate that heterologous expression of plant MT isoforms can enhance the tolerance of S. cerevisiae to multiple stresses.

  20. PrPC Undergoes Basal to Apical Transcytosis in Polarized Epithelial MDCK Cells

    PubMed Central

    Arkhipenko, Alexander; Syan, Sylvie; Victoria, Guiliana Soraya

    2016-01-01

    The Prion Protein (PrP) is an ubiquitously expressed glycosylated membrane protein attached to the external leaflet of the plasma membrane via a glycosylphosphatidylinositol anchor (GPI). While the misfolded PrPSc scrapie isoform is the infectious agent of prion disease, the cellular isoform (PrPC) is an enigmatic protein with unclear function. Of interest, PrP localization in polarized MDCK cells is controversial and its mechanism of trafficking is not clear. Here we investigated PrP traffic in MDCK cells polarized on filters and in three-dimensional MDCK cysts, a more physiological model of polarized epithelia. We found that, unlike other GPI-anchored proteins (GPI-APs), PrP undergoes basolateral-to-apical transcytosis in fully polarized MDCK cells. Following this event full-length PrP and its cleavage fragments are segregated in different domains of the plasma membrane in polarized cells in both 2D and 3D cultures. PMID:27389581

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

    PubMed Central

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

    2007-01-01

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

  2. Expression of multiple cbb3 cytochrome c oxidase isoforms by combinations of multiple isosubunits in Pseudomonas aeruginosa.

    PubMed

    Hirai, Takehiro; Osamura, Tatsuya; Ishii, Masaharu; Arai, Hiroyuki

    2016-10-24

    The ubiquitous opportunistic human pathogen Pseudomonas aeruginosa has five terminal oxidases for aerobic respiration and uses them under different growth conditions. Two of them are cbb3-type cytochrome c oxidases encoded by the gene clusters ccoN1O1Q1P1 and ccoN2O2Q2P2, which are the main terminal oxidases under high- and low-oxygen conditions, respectively. P. aeruginosa also has two orphan gene clusters, ccoN3Q3 and ccoN4Q4, encoding the core catalytic CcoN isosubunits, but the roles of these genes have not been clarified. We found that 16 active cbb3 isoforms could be produced by combinations of four CcoN, two CcoO, and two CcoP isosubunits. The CcoN3- or CcoN4-containing isoforms were produced in the WT cell membrane in response to nitrite and cyanide, respectively. The strains carrying these isoforms were more resistant to nitrite or cyanide under low-oxygen conditions. These results indicate that P. aeruginosa gains resistance to respiratory inhibitors using multiple cbb3 isoforms with different features, which are produced through exchanges of multiple core catalytic isosubunits.

  3. Selective upregulation of the expression of plasma membrane calcium ATPase isoforms upon differentiation and 1,25(OH)2D3-vitamin treatment of colon cancer cells.

    PubMed

    Ribiczey, Polett; Papp, Béla; Homolya, László; Enyedi, Ágnes; Kovács, Tünde

    2015-08-14

    We have previously presented co-expression of the plasma membrane calcium ATPase isoforms 4b (PMCA4b) and 1b (PMCA1b) in colon carcinoma cells, and selective upregulation of PMCA4b during differentiation initiated by short chain fatty acids or post-confluent growth. Here we show that the induction of PMCA4b expression is a characteristic feature of the post-confluency-induced differentiation of both enterocyte-type and goblet cell-type colon cancer cells. Vitamin D3 (1,25(OH)2D3) is a well-known regulator of intestinal Ca(2+) absorption and of basic cell functions such as growth and differentiation in various cell types. As PMCA proteins are involved both in intestinal Ca(2+) absorption and adenocarcinoma cell differentiation, we investigated the effect of 1,25(OH)2D3 on PMCA expression in enterocyte-like colon carcinoma cells, and monitored its effect on the expression of various differentiation markers. 1,25(OH)2D3 stimulated PMCA1b, but not PMCA4b expression without modulating the expression of the majority of the differentiation markers examined. Caco-2 cells differentiated in post-confluent cultures present normal enterocyte-like intestinal epithelial phenotype. To better understand the role of PMCA proteins in vectorial Ca(2+) transport by enterocytes, we also studied their subcellular localization in mature polarized Caco-2 cells. Both PMCA isoforms were located to the basolateral membrane, and the PMCA-specific immunofluorescent signal was significantly higher in vitamin D3-treated cells, underlining the 1,25(OH)2D3-induced upregulation of PMCA (presumably 1b isoform) expression in differentiated Caco-2 cells. We suggest that while PMCA1b has a housekeeping function in colon cancer cells, PMCA4b participates in the reorganization of the Ca(2+) signalling machinery during cell differentiation. The subcellular localization of PMCA1b and its selective 1,25(OH)2D3-dependent upregulation indicate that this isoform may have a specific role in 1,25(OH)2D3

  4. Cholesteryl ester loading of mouse peritoneal macrophages is associated with changes in the expression or modification of specific cellular proteins, including increase in an alpha-enolase isoform.

    PubMed

    Bottalico, L A; Kendrick, N C; Keller, A; Li, Y; Tabas, I

    1993-02-01

    This report explores the hypothesis that massive cholesteryl ester (CE) accumulation in macrophages, such as that occurring in atheroma foam cells, results in changes in the expression or modification of specific cellular proteins. Two-dimensional (2-D) gel electrophoretic patterns of metabolically labeled cellular proteins from mouse peritoneal macrophages that were loaded with CE (through incubation with acetylated low density lipoprotein [acetyl-LDL] for 4 days) were compared with those of control macrophages. Densitometric analysis of 2-D gel autoradiograms from the cell lysates revealed statistically significant changes in seven cellular proteins (five decreases and two increases). The changes in protein expression (foam cell versus control) ranged from a 458 +/- 164% (p < 0.001) increase to a 35 +/- 34% (p < 0.001) decrease (n = 11). Incubation of macrophages with beta-very low density lipoprotein, which also increased the CE content of macrophages (albeit to a lesser extent than acetyl-LDL), resulted in changes in five of the seven proteins. In contrast, incubation of cells with LDL, fucoidan, or latex beads, none of which caused CE accumulation, did not lead to significant changes in four of these five proteins. One of these four proteins, which increased fourfold to fivefold in foam cells (M(r) = 49,000; isoelectric point of 6.8), was purified by preparative 2-D gel electrophoresis. Internal amino acid sequence of cyanogen bromide fragments of this protein as well as Western blot analysis identified this protein as an isoform of alpha-enolase. The increased expression of this alpha-enolase isoform, which was seen as early as day 2 of acetyl-LDL incubation of the macrophages, was diminished by including an inhibitor of cholesterol esterification during the acetyl-LDL incubation period. In conclusion, macrophage foam cell formation is associated with distinct changes in protein expression, including a marked increase in an isoform of alpha

  5. Role of noradrenaline on the expression of the Na+/K+-ATPase alpha2 isoform and the contractility of cultured rat vas deferens.

    PubMed

    Quintas, Luis Eduardo M; Lafayette, Simone S L; Caricati-Neto, Afonso; Jurkiewicz, Aron; Noël, François

    2002-11-15

    Rat vasa deferentia were cultured for 3 days in Dulbecco's modified Eagle's medium in the absence or presence of 1 microM noradrenaline (NA) to investigate if the lack of NA release is the key factor to explain the selective reduction of the Na(+)/K(+)-ATPase alpha(2) isoform previously observed after in vivo denervation of this organ (Quintas et al., Biochem Pharmacol 2000;60:741-7). The lack of effects of the indirect sympathomimetic tyramine and the neuronal amine uptake blocker cocaine on NA curves indicated that cultured organs were denervated completely. Organ culture induced supersensitivity, expressed as a 6.3-fold increase of pD(2) and a 42% elevation of maximal contraction for NA but not for Ba(2+). Western blotting indicated that the level of the alpha(1) isoform of Na(+)/K(+)-ATPase was unchanged after organ culture, but the alpha(2) isoform was down-regulated drastically to levels that were barely detectable. The addition of NA to the culture medium did not prevent the reduction of alpha(2) expression although it did impede NA supersensitivity (in fact a 4-fold decrease of pD(2) and a 32% reduction of maximal response were observed after incubation in the presence of NA). A striking reduction of L-type Ca(2+) channel expression also was observed, indicated by an 85% decrease of [3H]isradipine binding sites. These data suggest that NA is a trophic factor relevant to the control of muscle contraction, mediated by alpha(1)-adrenoceptors, but not to the expression of either Na(+)/K(+)-ATPase or the L-type Ca(2+) channel.

  6. Expression of the human isoform of glutamate dehydrogenase, hGDH2, augments TCA cycle capacity and oxidative metabolism of glutamate during glucose deprivation in astrocytes.

    PubMed

    Nissen, Jakob D; Lykke, Kasper; Bryk, Jaroslaw; Stridh, Malin H; Zaganas, Ioannis; Skytt, Dorte M; Schousboe, Arne; Bak, Lasse K; Enard, Wolfgang; Pääbo, Svante; Waagepetersen, Helle S

    2017-03-01

    A key enzyme in brain glutamate homeostasis is glutamate dehydrogenase (GDH) which links carbohydrate and amino acid metabolism mediating glutamate degradation to CO2 and expanding tricarboxylic acid (TCA) cycle capacity with intermediates, i.e. anaplerosis. Humans express two GDH isoforms, GDH1 and 2, whereas most other mammals express only GDH1. hGDH1 is widely expressed in human brain while hGDH2 is confined to astrocytes. The two isoforms display different enzymatic properties and the nature of these supports that hGDH2 expression in astrocytes potentially increases glutamate oxidation and supports the TCA cycle during energy-demanding processes such as high intensity glutamatergic signaling. However, little is known about how expression of hGDH2 affects the handling of glutamate and TCA cycle metabolism in astrocytes. Therefore, we cultured astrocytes from cerebral cortical tissue of hGDH2-expressing transgenic mice. We measured glutamate uptake and metabolism using [(3) H]glutamate, while the effect on metabolic pathways of glutamate and glucose was evaluated by use of (13) C and (14) C substrates and analysis by mass spectrometry and determination of radioactively labeled metabolites including CO2 , respectively. We conclude that hGDH2 expression increases capacity for uptake and oxidative metabolism of glutamate, particularly during increased workload and aglycemia. Additionally, hGDH2 expression increased utilization of branched-chain amino acids (BCAA) during aglycemia and caused a general decrease in oxidative glucose metabolism. We speculate, that expression of hGDH2 allows astrocytes to spare glucose and utilize BCAAs during substrate shortages. These findings support the proposed role of hGDH2 in astrocytes as an important fail-safe during situations of intense glutamatergic activity. GLIA 2017;65:474-488.

  7. Drosophila HP1c isoform interacts with the zinc-finger proteins WOC and Relative-of-WOC to regulate gene expression.

    PubMed

    Font-Burgada, Joan; Rossell, David; Auer, Herbert; Azorín, Fernando

    2008-11-01

    Heterochromatin protein 1 (HP1) proteins are conserved in eukaryotes, with most species containing several isoforms. Based on the properties of Drosophila HP1a, it was proposed that HP1s bind H3K9me2,3 and recruit factors involved in heterochromatin assembly and silencing. Yet, it is unclear whether this general picture applies to all HP1 isoforms and functional contexts. Here, we report that Drosophila HP1c regulates gene expression, as (1) it localizes to active chromatin domains, where it extensively colocalizes with the poised form of RNApolymerase II (RNApol II), Pol IIo(ser5), and H3K4me3, suggesting a contribution to transcriptional regulation; (2) its targeting to a reporter gene does not induce silencing but, on the contrary, increases its expression, and (3) it interacts with the zinc-finger proteins WOC (without children) and Relative-of-WOC (ROW), which are putative transcription factors. Here, we also show that, although HP1c efficiently binds H3K9me2,3 in vitro, its binding to chromatin strictly depends on both WOC and ROW. Moreover, expression profiling indicates that HP1c, WOC, and ROW regulate a common gene expression program that, in part, is executed in the context of the nervous system. From this study, which unveils the essential contribution of DNA-binding proteins to HP1c functionality and recruitment, HP1 proteins emerge as an increasingly diverse family of chromatin regulators.

  8. RORγt, a Novel Isoform of an Orphan Receptor, Negatively Regulates Fas Ligand Expression and IL-2 Production in T Cells

    PubMed Central

    He, You-Wen; Deftos, Michael L.; Ojala, Ethan W.; Bevan, Michael J.

    2009-01-01

    Summary We have identified RORγt, a novel, thymus-specific isoform of the orphan nuclear receptor RORγ that is expressed predominantly in CD4+ CD8+ double-positive thymocytes. Ectopic expression of RORγt protects T cell hybridomas from activation-induced cell death by inhibiting the upregulation of Fas ligand. Following hybridoma stimulation, RORγt also inhibits IL-2 production but does not affect the induction of Nur-77 and Egr-3 nor the upregulation of CD69. Both the ligand-binding and DNA-binding domains of RORγt are required for this effect. We propose that the role of RORγt expression in immature thymocytes is to inhibit Fas ligand expression and cytokine secretion following engagement of their TCR during positive or negative selection. PMID:9881970

  9. Inference of Isoforms from Short Sequence Reads

    NASA Astrophysics Data System (ADS)

    Feng, Jianxing; Li, Wei; Jiang, Tao

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

  10. Carbonic Anhydrase III Is Expressed in Mouse Skeletal Muscles Independent of Fiber Type-Specific Myofilament Protein Isoforms and Plays a Role in Fatigue Resistance

    PubMed Central

    Feng, Han-Zhong; Jin, J.-P.

    2016-01-01

    Carbonic anhydrase III (CAIII) is a metabolic enzyme and a regulator for intracellular pH. CAIII has been reported with high level expression in slow twitch skeletal muscles. Here we demonstrate that CAIII is expressed in multiple slow and fast twitch muscles of adult mouse independent of the expression of myosin isoforms. Expressing similar fast type of myofilament proteins, CAIII-positive tibial anterior (TA) muscle exhibits higher tolerance to fatigue than that of CAIII-negative fast twitch extensor digitorum longus (EDL) muscle in in situ contractility studies. We further studied the muscles of CAIII knockout (Car3-KO) mice. The loss of CAIII in soleus and TA muscles in Car3-KO mice did not change muscle mass, sarcomere protein isoform contents, and the baseline twitch and tetanic contractility as compared with age-matched wild type (WT) controls. On the other hand, Car3-KO TA muscle showed faster force reduction at the beginning but higher resistance at the end during a fatigue test, followed by slower post fatigue recovery than that of WT TA muscle. Superfused Car3-KO soleus muscle also had faster total force reduction during fatigue test than that of WT soleus. However, it showed a less elevation of resting tension followed by a better post fatigue recovery under acidotic stress. CAIII was detected in neonatal TA and EDL muscle, downregulated during development, and then re-expressed in adult TA but not EDL muscles. The expression of CAIII in Tnnt1-KO myopathy mouse soleus muscle that has diminished slow fiber contents due to the loss of slow troponin T remained high. Car3-KO EDL, TA, and soleus muscles showed no change in the expression of mitochondria biomarker proteins. The data suggest a fiber type independent expression of CAIII with a role in the regulation of intracellular pH in skeletal muscle and may be explored as a target for improving fatigue resistance and for the treatment of TNNT1 myopathies. PMID:28018233

  11. Carbonic Anhydrase III Is Expressed in Mouse Skeletal Muscles Independent of Fiber Type-Specific Myofilament Protein Isoforms and Plays a Role in Fatigue Resistance.

    PubMed

    Feng, Han-Zhong; Jin, J-P

    2016-01-01

    Carbonic anhydrase III (CAIII) is a metabolic enzyme and a regulator for intracellular pH. CAIII has been reported with high level expression in slow twitch skeletal muscles. Here we demonstrate that CAIII is expressed in multiple slow and fast twitch muscles of adult mouse independent of the expression of myosin isoforms. Expressing similar fast type of myofilament proteins, CAIII-positive tibial anterior (TA) muscle exhibits higher tolerance to fatigue than that of CAIII-negative fast twitch extensor digitorum longus (EDL) muscle in in situ contractility studies. We further studied the muscles of CAIII knockout (Car3-KO) mice. The loss of CAIII in soleus and TA muscles in Car3-KO mice did not change muscle mass, sarcomere protein isoform contents, and the baseline twitch and tetanic contractility as compared with age-matched wild type (WT) controls. On the other hand, Car3-KO TA muscle showed faster force reduction at the beginning but higher resistance at the end during a fatigue test, followed by slower post fatigue recovery than that of WT TA muscle. Superfused Car3-KO soleus muscle also had faster total force reduction during fatigue test than that of WT soleus. However, it showed a less elevation of resting tension followed by a better post fatigue recovery under acidotic stress. CAIII was detected in neonatal TA and EDL muscle, downregulated during development, and then re-expressed in adult TA but not EDL muscles. The expression of CAIII in Tnnt1-KO myopathy mouse soleus muscle that has diminished slow fiber contents due to the loss of slow troponin T remained high. Car3-KO EDL, TA, and soleus muscles showed no change in the expression of mitochondria biomarker proteins. The data suggest a fiber type independent expression of CAIII with a role in the regulation of intracellular pH in skeletal muscle and may be explored as a target for improving fatigue resistance and for the treatment of TNNT1 myopathies.

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

    PubMed

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

    2012-07-01

    Peripheral activation of corticotropin-releasing factor receptor type 2 (CRF(2)) by urocortin 1, 2, or 3 (Ucns) exerts powerful effects on gastric function; however, little is known about their expression and regulation in the stomach. We investigated the expression of Ucns and CRF(2) isoforms by RT-PCR in the gastric corpus (GC) mucosa and submucosa plus muscle (S+M) or laser captured layers in naive rats, their regulations by lipopolysaccharide (LPS, 100 μg/kg ip) over 24 h, and the effect of the CRF(2) antagonist astresssin(2)-B (100 μg/kg sc) on LPS-induced delayed gastric emptying (GE) 2-h postinjection. Transcripts of Ucns and CRF(2b,) the most common wild-type CRF(2) isoform in the periphery, were expressed in all layers, including myenteric neurons. LPS increased Ucn mRNA levels significantly in both mucosa and S+M, reaching a maximal response at 6 h postinjection and returning to basal levels at 24 h except for Ucn 1 in S+M. By contrast, CRF(2b) mRNA level was significantly decreased in the mucosa and M+S with a nadir at 6 h. In addition, CRF(2a), reportedly only found in the brain, and the novel splice variant CRF(2a-3) were also detected in the GC, antrum, and pylorus. LPS reciprocally regulated these variants with a decrease of CRF(2a) and an increase of CRF(2a-3) in the GC 6 h postinjection. Astressin(2)-B exacerbated LPS-delayed GE (42-73%, P < 0.001). These data indicate that Ucn and CRF(2) isoforms are widely distributed throughout the rat stomach and inversely regulated by immune stress. The CRF(2) signaling system may act to counteract the early gastric motor alterations to endotoxemia.

  13. Expression of Total Vascular Endothelial Growth Factor and the Anti-angiogenic VEGF165b Isoform in the Vitreous of Patients with Retinopathy of Prematurity

    PubMed Central

    Zhao, Min; Xie, Wan-Kun; Bai, Yu-Jing; Huang, Lyu-Zhen; Wang, Bin; Liang, Jian-Hong; Yin, Hong; Li, Xiao-Xin; Shi, Xuan

    2015-01-01

    Background: This study was to examine the expression of total vascular endothelial growth factor (VEGF) and the anti-angiogenic VEGF165b isoform in the vitreous body of retinopathy of prematurity (ROP) patients, and to further study the role of the VEGF splicing in the development of ROP. Methods: This was a prospective clinical laboratory investigation study. All patients enrolled received standard ophthalmic examination with stage 4 ROP that required vitrectomy to collect the vitreous samples. The control samples were from congenital cataract patients. The expression of total VEGF and the anti-angiogenic VEGF165b were measured by enzyme-linked immunosorbent assay. Results were analyzed statistically using nonparametric tests. Results: The total VEGF level was markedly elevated in ROP samples while VEGF165b was markedly decreased compared to control group. The relative protein expression level of VEGF165b isoform was significantly decreased in ROP patients which were correlated with the ischemia-induced neovascularization. Conclusions: There was a switch of VEGF splicing from anti-angiogenic to pro-angiogenic family in ROP patients. A specific inhibitor that more selectively targets VEGF165and controls the VEGF splicing between pro- and anti-angiogenic families might be a more effective therapy for ROP. PMID:26365970

  14. Effects of chronic academic stress on mental state and expression of glucocorticoid receptor α and β isoforms in healthy Japanese medical students.

    PubMed

    Kurokawa, Ken; Tanahashi, Toshihito; Murata, Akiho; Akaike, Yoko; Katsuura, Sakurako; Nishida, Kensei; Masuda, Kiyoshi; Kuwano, Yuki; Kawai, Tomoko; Rokutan, Kazuhito

    2011-07-01

    Chronic academic stress responses were assessed by measuring mental state, salivary cortisol levels, and the glucocorticoid receptor (GR) gene expression in healthy Japanese medical students challenging the national medical license examination. Mental states of 17 male and 9 female medical undergraduates, aged 25.0 ± 1.2 years (mean ± SD), were assessed by the State and Trait Anxiety Inventory (STAI) and the Self-Rating Depression Scale (SDS) 2 months before, 2 days before, and 1 month after the examination. At the same time points, saliva and blood were collected. STAI-state scores peaked 2 days before the examination. Scores on STAI-trait and SDS, and salivary cortisol levels were consistently higher during the pre-examination period. One month after the examination, all these measures had significantly decreased to baseline levels. Real-time reverse transcription PCR showed that this chronic anxious state did not change the expression of the functional GRα mRNA isoform in peripheral leukocytes, while it resulted in reduced expression of the GRβ isoform 2 days before the examination. Our results replicate and extend a significant impact of chronic academic stressors on the mental state of healthy Japanese medical students and suggest a possible association of GRβ gene in response to psychological stress.

  15. Cellular expression of isoforms of endothelin-converting enzyme-1 (ECE-1c, ECE-1b and ECE-1a) and endothelin-converting enzyme-2.

    PubMed

    Davenport, A P; Kuc, R E

    2000-11-01

    Our aim was to compare the cellular expression of endothelin-converting enzyme-1 (ECE-1) isoforms and ECE-2 using immunocytochemistry in normal and diseased human tissue. Intense ECE-1b immunoreactivity was present within renal and pulmonary epithelial cells with lower levels of staining displayed by ECE-1c, ECE-1a and ECE-2 antisera. Staining was detected with all antisera (except ECE-1a) within the endothelium of renal and pulmonary vessels having a range of lumen diameters as well as pial arteries and intracerebral vessels penetrating brain. ECE-1b, ECE-1c and ECE-2 immunoreactivity was localized to perivascular astrocytes and neuronal processes in the cerebral cortex. In diseased vessels, ECE-1c, ECE-1b and ECE-2 antisera stained macrophages infiltrating atherosclerotic plaques within coronary arteries. These results suggest ECE-1b and ECE-2 may be widely expressed in normal tissue from humans and inhibition of ECE-1 isoforms and ECE-2 expressed by cells such as macrophages in pathophysiological tissue may be an additional therapeutic target in cardiovascular disease.

  16. Polar substitutions in helix 3 of the prion protein produce transmembrane isoforms that disturb vesicle trafficking

    PubMed Central

    Sanchez-Garcia, Jonatan; Arbelaez, Daniela; Jensen, Kurt; Rincon-Limas, Diego E.; Fernandez-Funez, Pedro

    2013-01-01

    Prion diseases encompass a diverse group of neurodegenerative conditions characterized by the accumulation of misfolded prion protein (PrP) isoforms. Other conformational variants of PrP have also been proposed to contribute to neurotoxicity in prion diseases, including misfolded intermediates as well as cytosolic and transmembrane isoforms. To better understand PrP neurotoxicity, we analyzed the role of two highly conserved methionines in helix 3 on PrP biogenesis, folding and pathogenesis. Expression of the PrP-M205S and -M205,212S mutants in Drosophila led to hyperglycosylation, intracellular accumulation and widespread conformational changes due to failure of oxidative folding. Surprisingly, PrP-M205S and -M205,212S acquired a transmembrane topology (Ctm) previously linked to mutations in the signal peptide (SP) and the transmembrane domain (TMD). PrP-M205,212S also disrupted the accumulation of key neurodevelopmental proteins in lipid rafts, resulting in shortened axonal projections. These results uncover a new role for the hydrophobic domain in promoting oxidative folding and preventing the formation of neurotoxic Ctm PrP, mechanisms that may be relevant in the pathogenesis of both inherited and sporadic prion diseases. PMID:23771030

  17. Polar substitutions in helix 3 of the prion protein produce transmembrane isoforms that disturb vesicle trafficking.

    PubMed

    Sanchez-Garcia, Jonatan; Arbelaez, Daniela; Jensen, Kurt; Rincon-Limas, Diego E; Fernandez-Funez, Pedro

    2013-11-01

    Prion diseases encompass a diverse group of neurodegenerative conditions characterized by the accumulation of misfolded prion protein (PrP) isoforms. Other conformational variants of PrP have also been proposed to contribute to neurotoxicity in prion diseases, including misfolded intermediates as well as cytosolic and transmembrane isoforms. To better understand PrP neurotoxicity, we analyzed the role of two highly conserved methionines in helix 3 on PrP biogenesis, folding and pathogenesis. Expression of the PrP-M205S and -M205,212S mutants in Drosophila led to hyperglycosylation, intracellular accumulation and widespread conformational changes due to failure of oxidative folding. Surprisingly, PrP-M205S and -M205,212S acquired a transmembrane topology (Ctm) previously linked to mutations in the signal peptide (SP) and the transmembrane domain (TMD). PrP-M205,212S also disrupted the accumulation of key neurodevelopmental proteins in lipid rafts, resulting in shortened axonal projections. These results uncover a new role for the hydrophobic domain in promoting oxidative folding and preventing the formation of neurotoxic Ctm PrP, mechanisms that may be relevant in the pathogenesis of both inherited and sporadic prion diseases.

  18. Molecular properties and fibril ultrastructure of types II and XI collagens in cartilage of mice expressing exclusively the α1(IIA) collagen isoform.

    PubMed

    McAlinden, Audrey; Traeger, Geoffrey; Hansen, Uwe; Weis, Mary Ann; Ravindran, Soumya; Wirthlin, Louisa; Eyre, David R; Fernandes, Russell J

    2014-02-01

    Until now, no biological tools have been available to determine if a cross-linked collagen fibrillar network derived entirely from type IIA procollagen isoforms, can form in the extracellular matrix (ECM) of cartilage. Recently, homozygous knock-in transgenic mice (Col2a1(+ex2), ki/ki) were generated that exclusively express the IIA procollagen isoform during post-natal development while type IIB procollagen, normally present in the ECM of wild type mice, is absent. The difference between these Col2a1 isoforms is the inclusion (IIA) or exclusion (IIB) of exon 2 that is alternatively spliced in a developmentally regulated manner. Specifically, chondroprogenitor cells synthesize predominantly IIA mRNA isoforms while differentiated chondrocytes produce mainly IIB mRNA isoforms. Recent characterization of the Col2a1(+ex2) mice has surprisingly shown that disruption of alternative splicing does not affect overt cartilage formation. In the present study, biochemical analyses showed that type IIA collagen extracted from ki/ki mouse rib cartilage can form homopolymers that are stabilized predominantly by hydroxylysyl pyridinoline (HP) cross-links at levels that differed from wild type rib cartilage. The findings indicate that mature type II collagen derived exclusively from type IIA procollagen molecules can form hetero-fibrils with type XI collagen and contribute to cartilage structure and function. Heteropolymers with type XI collagen also formed. Electron microscopy revealed mainly thin type IIA collagen fibrils in ki/ki mouse rib cartilage. Immunoprecipitation and mass spectrometry of purified type XI collagen revealed a heterotrimeric molecular composition of α1(XI)α2(XI)α1(IIA) chains where the α1(IIA) chain is the IIA form of the α3(XI) chain. Since the N-propeptide of type XI collagen regulates type II collagen fibril diameter in cartilage, the retention of the exon 2-encoded IIA globular domain would structurally alter the N-propeptide of type XI collagen

  19. Akt isoforms in vascular disease

    PubMed Central

    Yu, Haixiang; Littlewood, Trevor; Bennett, Martin

    2015-01-01

    The mammalian serine/threonine Akt kinases comprise three closely related isoforms: Akt1, Akt2 and Akt3. Akt activation has been implicated in both normal and disease processes, including in development and metabolism, as well as cancer and cardiovascular disease. Although Akt signalling has been identified as a promising therapeutic target in cancer, its role in cardiovascular disease is less clear. Importantly, accumulating evidence suggests that the three Akt isoforms exhibit distinct tissue expression profiles, localise to different subcellular compartments, and have unique modes of activation. Consistent with in vitro findings, genetic studies in mice show distinct effects of individual Akt isoforms on the pathophysiology of cardiovascular disease. This review summarises recent studies of individual Akt isoforms in atherosclerosis, vascular remodelling and aneurysm formation, to provide a comprehensive overview of Akt function in vascular disease. PMID:25929188

  20. Alternative Isoform Analysis of Ttc8 Expression in the Rat Pineal Gland Using a Multi-Platform Sequencing Approach Reveals Neural Regulation.

    PubMed

    Hartley, Stephen W; Mullikin, James C; Klein, David C; Park, Morgan; Coon, Steven L

    Alternative isoform regulation (AIR) vastly increases transcriptome diversity and plays an important role in numerous biological processes and pathologies. However, the detection and analysis of isoform-level differential regulation is difficult, particularly in the face of complex and incompletely-annotated transcriptomes. Here we have used Illumina short-read/high-throughput RNA-Seq to identify 55 genes that exhibit neurally-regulated AIR in the pineal gland, and then used two other complementary experimental platforms to further study and characterize the Ttc8 gene, which is involved in Bardet-Biedl syndrome and non-syndromic retinitis pigmentosa. Use of the JunctionSeq analysis tool led to the detection of several novel exons and splice junctions in this gene, including two novel alternative transcription start sites which were found to display disproportionately strong neurally-regulated differential expression in several independent experiments. These high-throughput sequencing results were validated and augmented via targeted qPCR and long-read Pacific Biosciences SMRT sequencing. We confirmed the existence of numerous novel splice junctions and the selective upregulation of the two novel start sites. In addition, we identified more than 20 novel isoforms of the Ttc8 gene that are co-expressed in this tissue. By using information from multiple independent platforms we not only greatly reduce the risk of errors, biases, and artifacts influencing our results, we also are able to characterize the regulation and splicing of the Ttc8 gene more deeply and more precisely than would be possible via any single platform. The hybrid method outlined here represents a powerful strategy in the study of the transcriptome.

  1. Alternative Isoform Analysis of Ttc8 Expression in the Rat Pineal Gland Using a Multi-Platform Sequencing Approach Reveals Neural Regulation

    PubMed Central

    Mullikin, James C.; Klein, David C.; Park, Morgan; Coon, Steven L.

    2016-01-01

    Alternative isoform regulation (AIR) vastly increases transcriptome diversity and plays an important role in numerous biological processes and pathologies. However, the detection and analysis of isoform-level differential regulation is difficult, particularly in the face of complex and incompletely-annotated transcriptomes. Here we have used Illumina short-read/high-throughput RNA-Seq to identify 55 genes that exhibit neurally-regulated AIR in the pineal gland, and then used two other complementary experimental platforms to further study and characterize the Ttc8 gene, which is involved in Bardet-Biedl syndrome and non-syndromic retinitis pigmentosa. Use of the JunctionSeq analysis tool led to the detection of several novel exons and splice junctions in this gene, including two novel alternative transcription start sites which were found to display disproportionately strong neurally-regulated differential expression in several independent experiments. These high-throughput sequencing results were validated and augmented via targeted qPCR and long-read Pacific Biosciences SMRT sequencing. We confirmed the existence of numerous novel splice junctions and the selective upregulation of the two novel start sites. In addition, we identified more than 20 novel isoforms of the Ttc8 gene that are co-expressed in this tissue. By using information from multiple independent platforms we not only greatly reduce the risk of errors, biases, and artifacts influencing our results, we also are able to characterize the regulation and splicing of the Ttc8 gene more deeply and more precisely than would be possible via any single platform. The hybrid method outlined here represents a powerful strategy in the study of the transcriptome. PMID:27684375

  2. In vitro expression of the alpha-smooth muscle actin isoform by rat lung mesenchymal cells: regulation by culture condition and transforming growth factor-beta.

    PubMed

    Mitchell, J J; Woodcock-Mitchell, J L; Perry, L; Zhao, J; Low, R B; Baldor, L; Absher, P M

    1993-07-01

    alpha-Smooth muscle actin (alpha SM actin)-containing cells recently have been demonstrated in intraalveolar lesions in both rat and human tissues following lung injury. In order to develop model systems for the study of such cells, we examined cultured lung cell lines for this phenotype. The adult rat lung fibroblast-like "RL" cell lines were found to express alpha SM actin mRNA and protein and to organize this actin into stress fiber-like structures. Immunocytochemical staining of subclones of the RL87 line demonstrated the presence in the cultures of at least four cell phenotypes, one that fails to express alpha SM actin and three distinct morphologic types that do express alpha SM actin. The proportion of cellular actin that is the alpha-isoform was modulated by the culture conditions. RL cells growing at low density expressed minimal alpha SM actin. On reaching confluent densities, however, alpha SM actin increased to at least 20% of the total actin content. This effect, combined with the observation that the most immunoreactive cells were those that displayed overlapping cell processes in culture, suggests that cell-cell contact may be involved in actin isoform regulation in these cells. Similar to the response of some smooth muscle cell lines, alpha SM actin expression in RL cells also was promoted by conditions, e.g., maintenance in low serum medium, which minimize cell division. alpha SM actin expression was modulated in RL cells by the growth factor transforming growth factor-beta. Addition of this cytokine to growing cells substantially elevated the proportion of alpha SM actin protein.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Molecular Cloning and Expression Analysis of hyp-1 Type PR-10 Family Genes in Hypericum perforatum.

    PubMed

    Karppinen, Katja; Derzsó, Emese; Jaakola, Laura; Hohtola, Anja

    2016-01-01

    Hypericum perforatum L. is an important medicinal plant for the treatment of depression. The plant contains bioactive hypericins that accumulate in dark glands present especially in reproductive parts of the plant. In this study, pathogenesis-related class 10 (PR-10) family genes were identified in H. perforatum, including three previously unidentified members with sequence homology to hyp-1, a phenolic coupling protein that has earlier been suggested to participate in biosynthesis and binding/transportation of hypericin. The PR-10 genes showed constitutive but variable expression patterns in different H. perforatum tissues. They were all expressed at relatively high levels in leaves, variably in roots and low levels in stem and reproductive parts of the plant with no specific association with dark glands. The gene expression was up-regulated in leaves after salicylic acid, abscisic acid and wounding treatments but with variable levels. To study exact location of the gene expression, in situ hybridization of hyp-1 transcripts was performed and the accumulation of the Hyp-1 protein was examined in various tissues. The presence of Hyp-1 protein in H. perforatum tissues mostly paralleled with the mRNA levels. In situ RNA hybridization localized the hyp-1 transcripts predominantly in vascular tissues in root and stem, while in leaf the mRNA levels were high also in mesophyll cells in addition to vasculature. Our results indicate that the studied PR-10 genes are likely to contribute to the defense responses in H. perforatum. Furthermore, despite the location of the hyp-1 transcripts in vasculature, no support for the transportation of the Hyp-1 protein to dark glands was found in the current study. The present results together with earlier data question the role of the hyp-1 as a key gene responsible for the hypericin biosynthesis in dark glands of H. perforatum.

  4. Molecular Cloning and Expression Analysis of hyp-1 Type PR-10 Family Genes in Hypericum perforatum

    PubMed Central

    Karppinen, Katja; Derzsó, Emese; Jaakola, Laura; Hohtola, Anja

    2016-01-01

    Hypericum perforatum L. is an important medicinal plant for the treatment of depression. The plant contains bioactive hypericins that accumulate in dark glands present especially in reproductive parts of the plant. In this study, pathogenesis-related class 10 (PR-10) family genes were identified in H. perforatum, including three previously unidentified members with sequence homology to hyp-1, a phenolic coupling protein that has earlier been suggested to participate in biosynthesis and binding/transportation of hypericin. The PR-10 genes showed constitutive but variable expression patterns in different H. perforatum tissues. They were all expressed at relatively high levels in leaves, variably in roots and low levels in stem and reproductive parts of the plant with no specific association with dark glands. The gene expression was up-regulated in leaves after salicylic acid, abscisic acid and wounding treatments but with variable levels. To study exact location of the gene expression, in situ hybridization of hyp-1 transcripts was performed and the accumulation of the Hyp-1 protein was examined in various tissues. The presence of Hyp-1 protein in H. perforatum tissues mostly paralleled with the mRNA levels. In situ RNA hybridization localized the hyp-1 transcripts predominantly in vascular tissues in root and stem, while in leaf the mRNA levels were high also in mesophyll cells in addition to vasculature. Our results indicate that the studied PR-10 genes are likely to contribute to the defense responses in H. perforatum. Furthermore, despite the location of the hyp-1 transcripts in vasculature, no support for the transportation of the Hyp-1 protein to dark glands was found in the current study. The present results together with earlier data question the role of the hyp-1 as a key gene responsible for the hypericin biosynthesis in dark glands of H. perforatum. PMID:27148343

  5. Recombinant AAV-PR39-mediated hypoxia-inducible factor 1α gene expression attenuates myocardial infarction.

    PubMed

    Sun, Lijun; Hao, Yuewen; Nie, Xiaowei; Xu, Jian; Li, Zhenwu; Zhang, Wei; Liu, Ying; Zhang, Xuexin

    2014-01-01

    PR39 is an angiogenic masterswitch protein, belonging to the second generation of angiogenic growth factors. However, the role of recombinant adeno-associated virus (AAV) carrying the PR39 fusion gene (AAV-PR39) in acute myocardial infarction remains unclear. Therefore, in this study, we investigated the role of AAV-PR39 in an experimental animal model of acute myocardial infarction. The PR39 gene was fused with the transmembrane peptide, TAT, 6xHis‑tag and NT4 signal sequences. AAV-PR39 was then obtained by calcium phosphate co-precipitation. A total of 18 healthy Chinese mini pigs were randomly divided into an experimental groups (the AAV-PR39-treated group) and a control group [phosphated-buffered saline (PBS)-treated group]. Following the induction of myocardial infarction, enhanced 3.0T MR imaging was performed to observe the changes in myocardial signal intensity at 0 h, 1, 2 and 3 weeks. The expression of hypoxia-inducible factor‑1α (HIF-1α) in the myocardial tissues was determined by SABC immunohistochemistry. In addition, in vitro experiments using CRL-1730 endothelial cells transfected with AAV vector containing NT4-TAT-His-PR39 revealed that the AAV-PR39-treated group had a significantly higher expression of HIF-1α compared with the control group. Moreover, PR39 regulated the HIF-1α-induced expression of angiogenic growth factors. Under hypoxic conditions, the anti-apoptotic effects in the AAV-PR39 group were more pronounced than those observed in the control (PBS-treated) group. In vivo, the enforced expression of recombinant PR39 elevated the level of HIF-1α under hypoxic conditions and decreased the size of the infarcted areas by upregulating the expression of HIF-1α in the areas surrounding the infarct area. Taken together, our data demonstrate that the recombinant AAV-PR39-mediated HIF-1α expression attenuates myocardial infarction, indicating that AAV-PR39 may serve as a novel therapeutic agent for the treatment of myocardial infarction.

  6. Single-fiber expression and fiber-specific adaptability to short-term intense exercise training of Na+-K+-ATPase α- and β-isoforms in human skeletal muscle.

    PubMed

    Wyckelsma, V L; McKenna, M J; Serpiello, F R; Lamboley, C R; Aughey, R J; Stepto, N K; Bishop, D J; Murphy, R M

    2015-03-15

    The Na(+)-K(+)-ATPase (NKA) plays a key role in muscle excitability, but little is known in human skeletal muscle about fiber-type-specific differences in NKA isoform expression or adaptability. A vastus lateralis muscle biopsy was taken in 17 healthy young adults to contrast NKA isoform protein relative abundance between type I and IIa fibers. We further investigated muscle fiber-type-specific NKA adaptability in eight of these adults following 4-wk repeated-sprint exercise (RSE) training, comprising three sets of 5 × 4-s sprints, 3 days/wk. Single fibers were separated, and myosin heavy chain (I and IIa) and NKA (α1-3 and β1-3) isoform abundance were determined via Western blotting. All six NKA isoforms were expressed in both type I and IIa fibers. No differences between fiber types were found for α1-, α2-, α3-, β1-, or β3-isoform abundances. The NKA β2-isoform was 27% more abundant in type IIa than type I fibers (P < 0.05), with no other fiber-type-specific trends evident. RSE training increased β1 in type IIa fibers (pretraining 0.70 ± 0.25, posttraining 0.84 ± 0.24 arbitrary units, 42%, P < 0.05). No training effects were found for other NKA isoforms. Thus human skeletal muscle expresses all six NKA isoforms and not in a fiber-type-specific manner; this points to their different functional roles in skeletal muscle cells. Detection of elevated NKA β1 after RSE training demonstrates the sensitivity of the single-fiber Western blotting technique for fiber-type-specific intervention effects.

  7. Isoform composition and gene expression of thick and thin filament proteins in striated muscles of mice after 30-day space flight.

    PubMed

    Ulanova, Anna; Gritsyna, Yulia; Vikhlyantsev, Ivan; Salmov, Nikolay; Bobylev, Alexander; Abdusalamova, Zarema; Rogachevsky, Vadim; Shenkman, Boris; Podlubnaya, Zoya

    2015-01-01

    Changes in isoform composition, gene expression of titin and nebulin, and isoform composition of myosin heavy chains as well as changes in titin phosphorylation level in skeletal (m. gastrocnemius, m. tibialis anterior, and m. psoas) and cardiac muscles of mice were studied after a 30-day-long space flight onboard the Russian spacecraft "BION-M" number 1. A muscle fibre-type shift from slow-to-fast and a decrease in the content of titin and nebulin in the skeletal muscles of animals from "Flight" group was found. Using Pro-Q Diamond staining, an ~3-fold increase in the phosphorylation level of titin in m. gastrocnemius of mice from the "Flight" group was detected. The content of titin and its phosphorylation level in the cardiac muscle of mice from "Flight" and "Control" groups did not differ; nevertheless an increase (2.2 times) in titin gene expression in the myocardium of flight animals was found. The observed changes are discussed in the context of their role in the contractile activity of striated muscles of mice under conditions of weightlessness.

  8. Isoform Composition and Gene Expression of Thick and Thin Filament Proteins in Striated Muscles of Mice after 30-Day Space Flight

    PubMed Central

    Ulanova, Anna; Gritsyna, Yulia; Vikhlyantsev, Ivan; Salmov, Nikolay; Bobylev, Alexander; Abdusalamova, Zarema; Rogachevsky, Vadim; Shenkman, Boris; Podlubnaya, Zoya

    2015-01-01

    Changes in isoform composition, gene expression of titin and nebulin, and isoform composition of myosin heavy chains as well as changes in titin phosphorylation level in skeletal (m. gastrocnemius, m. tibialis anterior, and m. psoas) and cardiac muscles of mice were studied after a 30-day-long space flight onboard the Russian spacecraft “BION-M” number 1. A muscle fibre-type shift from slow-to-fast and a decrease in the content of titin and nebulin in the skeletal muscles of animals from “Flight” group was found. Using Pro-Q Diamond staining, an ~3-fold increase in the phosphorylation level of titin in m. gastrocnemius of mice from the “Flight” group was detected. The content of titin and its phosphorylation level in the cardiac muscle of mice from “Flight” and “Control” groups did not differ; nevertheless an increase (2.2 times) in titin gene expression in the myocardium of flight animals was found. The observed changes are discussed in the context of their role in the contractile activity of striated muscles of mice under conditions of weightlessness. PMID:25664316

  9. Different N-terminal isoforms of Oct-1 control expression of distinct sets of genes and their high levels in Namalwa Burkitt's lymphoma cells affect a wide range of cellular processes

    PubMed Central

    Pankratova, Elizaveta V.; Stepchenko, Alexander G.; Portseva, Tatiana; Mogila, Vladic A.; Georgieva, Sofia G.

    2016-01-01

    Oct-1 transcription factor has various functions in gene regulation. Its expression level is increased in several types of cancer and is associated with poor survival prognosis. Here we identified distinct Oct-1 protein isoforms in human cells and compared gene expression patterns and functions for Oct-1A, Oct-1L, and Oct-1X isoforms that differ by their N-terminal sequences. The longest isoform, Oct-1A, is abundantly expressed and is the main Oct-1 isoform in most of human tissues. The Oct-1L and the weakly expressed Oct-1X regulate the majority of Oct-1A targets as well as additional sets of genes. Oct-1X controls genes involved in DNA replication, DNA repair, RNA processing, and cellular response to stress. The high level of Oct-1 isoforms upregulates genes related to cell cycle progression and activates proliferation both in Namalwa Burkitt's lymphoma cells and primary human fibroblasts. It downregulates expression of genes related to antigen processing and presentation, cytokine-cytokine receptor interaction, oxidative metabolism, and cell adhesion, thus facilitating pro-oncogenic processes. PMID:27407111

  10. Frequent Missense and Insertion/Deletion Polymorphisms in the Ovine Shadoo Gene Parallel Species-Specific Variation in PrP

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: The cellular prion protein PrP**C is encoded by the Prnp gene. This protein is expressed in the central nervous system (CNS) and serves as a precursor to the misfolded PrP**S**c isoform in prion diseases. The prototype prion disease is scrapie in sheep, and, whereas, Prnp exhibits com...

  11. Enhanced expression of epithelial sodium channels causes salt-induced hypertension in mice through inhibition of the α2-isoform of Na+, K+-ATPase.

    PubMed

    Leenen, Frans H H; Hou, Xiaohong; Wang, Hong-Wei; Ahmad, Monir

    2015-05-01

    Knockout of the Nedd4-2 gene in mice results in overexpression of epithelial sodium channels (ENaC) on the plasma membrane in the kidney, choroid plexus and brain nuclei. These mice exhibit enhanced pressor responses to CSF [Na(+)] as well as dietary salt-induced hypertension which both can be blocked by central infusion of the ENaC blocker benzamil. Functional studies suggest that ENaC activation in the CNS results in release of endogenous ouabain (EO) and inhibition of the α2-isoform of Na(+), K(+)-ATPase. To test this concept more specifically, we studied Nedd4-2(-/-) mice expressing the ouabain-resistant α2R/R-isoform of Na(+), K(+)-ATPase. Intracerebroventricular (icv) infusion of Na(+)-rich aCSF (225 mmol/L Na(+) at 0.4 μL/min) increased MAP by 10-15 mmHg in wild-type mice and by 25-30 mmHg in Nedd4-2(-/-) mice, but by only ~5 mmHg in α2R/R and in α2R/R/Nedd4-2(-/-) mice. Icv infusion of EO-binding Fab fragments also blocked the BP response in Nedd4-2(-/-) mice. In Nedd4-2(-/-) mice, 8% high-salt diet increased MAP by 25-30 mmHg, but in α2R/R/Nedd4-2(-/-) mice, it increased by only 5-10 mmHg. In contrast, Nedd4-2(-/-) or α2R/R did not affect the hypertension caused by sc infusion of Ang II. These findings substantiate the concept that enhanced ENaC activity causes salt-induced pressor responses mainly through EO inhibiting the α2-isoform of Na(+), K(+)-ATPase in the brain.

  12. ICAM-1: isoforms and phenotypes.

    PubMed

    Ramos, Theresa N; Bullard, Daniel C; Barnum, Scott R

    2014-05-15

    ICAM-1 plays an important role in leukocyte trafficking, immunological synapse formation, and numerous cellular immune responses. Although considered a single glycoprotein, there are multiple membrane-bound and soluble ICAM-1 isoforms that arise from alternative splicing and proteolytic cleavage during inflammatory responses. The function and expression of these isoforms on various cell types are poorly understood. In the generation of ICAM-1-deficient mice, two isoform-deficient ICAM-1 mutants were inadvertently produced as a result of alternative splicing. These mice, along with true ICAM-1-deficient mice and newly generated ICAM-1-transgenic mice, have provided the opportunity to begin examining the role of ICAM-1 isoforms (singly or in combination) in various disease settings. In this review, we highlight the sharply contrasting disease phenotypes using ICAM-1 isoform mutant mice. These studies demonstrate that ICAM-1 immunobiology is highly complex but that individual isoforms, aside from the full-length molecule, make significant contributions to disease development and pathogenesis.

  13. ICAM-1: Isoforms and Phenotypes

    PubMed Central

    Ramos, Theresa N.; Bullard, Daniel C.; Barnum, Scott R.

    2014-01-01

    Intercellular adhesion molecule-1 (ICAM-1) plays an important role in leukocyte trafficking, immunological synapse formation and, numerous cellular immune responses. Although considered a single glycoprotein, there are multiple membrane bound and soluble ICAM-1 isoforms which arise from alternative splicing and proteolytic cleavage during inflammatory responses. The function and expression of these isoforms on various cell types is poorly understood. In the generation of ICAM-1-deficient mice, two isoform-deficient ICAM-1 mutants were inadvertently produced due to alternative splicing. These mice along with true ICAM-1-deficient mice and newly generated ICAM-1 transgenic mice have provided the opportunity to begin examining the role of ICAM-1 isoforms (singly or in combination) in various disease settings. In this review we highlight the sharply contrasting disease phenotypes using ICAM-1 isoform mutant mice. These studies demonstrate that ICAM-1 immunobiology is highly complex but that individual isoforms, aside from the full-length molecule, make significant contributions to disease development and pathogenesis. PMID:24795464

  14. Human adult chondrocytes express hepatocyte growth factor (HGF) isoforms but not HgF: potential implication of osteoblasts on the presence of HGF in cartilage.

    PubMed

    Guévremont, Melanie; Martel-Pelletier, Johanne; Massicotte, Frédéric; Tardif, Ginette; Pelletier, Jean-Pierre; Ranger, Pierre; Lajeunesse, Daniel; Reboul, Pascal

    2003-06-01

    HGF is increased in human OA cartilage, possibly from Ob's. RT-PCR shows HGF isoforms are differently regulated between chondrocytes and Ob. A paracrine cross-talk between subchondral bone and cartilage may occur during OA. Recently, hepatocyte growth factor (HGF) has been identified by immunohistochemistry in cartilage and more particularly in the deep zone of human osteoarthritic (OA) cartilage. By investigating HGF expression in cartilage, we found that chondrocytes did not express HGF; however, they expressed the two truncated isoforms, namely HGF/NK1 and HGF/NK2. Because the only other cells localized near the deep zone are osteoblasts from the subchondral bone plate, we hypothesized that they were expressing HGF. Indeed, we found that HGF was synthesized by osteoblasts from the subchondral bone plate. Moreover, OA osteoblasts produced five times more HGF than normal osteoblasts and almost no HGF/NK1, unlike normal osteoblasts. Because prostaglandin E2 (PGE2) and pro-inflammatory cytokines such as interleukin (IL)-1 and IL-6 are involved in OA progression, we investigated whether these factors impact HGF produced by normal osteoblasts. PGE2 was the only factor tested that was able to stimulate HGF synthesis. However, the addition of NS398, a selective inhibitor of cyclo-oxygenase-2 (COX-2) had no effect on HGF produced by OA osteoblasts. HGF/NK2 had a moderate stimulating effect on HGF production by normal osteoblasts, whereas osteocalcin was not modulated by either HGF or HGF/NK2. When investigating signaling routes that might be implicated in OA osteoblast-produced HGF, we found that protein kinase A was at least partially involved. In summary, this study raises the hypothesis that the HGF found in articular cartilage is produced by osteoblasts, diffuses into the cartilage, and may be implicated in the OA process.

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

    PubMed

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

    2015-08-01

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

  16. Luteotropic and luteolytic factors regulate mRNA and protein expression of progesterone receptor isoforms A and B in the bovine endometrium.

    PubMed

    Rekawiecki, Robert; Kowalik, Magdalena Karolina; Kotwica, Jan

    2014-12-17

    The aim of the present study was to examine the effects of luteotropic and luteolytic factors on the mRNA and protein levels of progesterone receptor isoforms A (PGRA) and B (PGRB) in the bovine endometrium. Endometrial slices from Days 6-10 and 17-20 of the oestrous cycle were treated with LH (100ngmL-1), oestradiol (E2; 1×10-8M), prostaglandin (PG) E2 (1×10-6M) and PGF2? (1×10-6M) and the nitric oxide donor NONOate (1×10-4M); these treatments lasted for 6h for mRNA expression analysis and 24h for protein expression analysis. On Days 6-10 of the oestrous cycle PGRAB (PGRAB; the entire PGRA mRNA sequence is common to the PGRB mRNA sequence) mRNA expression in endometrial slices was enhanced by E2 treatment (PPGRB mRNA expression was increased by LH (PPPPGRAB mRNA expression increased after E2 (P2 (PPGRB mRNA expression was increased by PGE2 (P2? (PPPPPP2? (P2 (P2? (P<0.001). These data suggest that luteotropic and luteolytic factors affect PGRA and PGRB mRNA and protein levels, and this may regulate the effects of progesterone on endometrial cells.

  17. Novel, primate-specific PDE10A isoform highlights gene expression complexity in human striatum with implications on the molecular pathology of bipolar disorder.

    PubMed

    MacMullen, C M; Vick, K; Pacifico, R; Fallahi-Sichani, M; Davis, R L

    2016-02-23

    Bipolar disorder is a highly heritable neuropsychiatric disorder affecting nearly 2.5% of the population. Prior genetic studies identified a panel of common and rare single-nucleotide polymorphisms associated with the disease that map to the first intron of the PDE10A gene. RNA sequencing of striatal brain tissue from bipolar and healthy control subjects identified a novel transcript of PDE10A, named PDE10A19, that codes for a PDE10A isoform with a unique N terminus. Genomic sequences that can encode the novel N terminus were conserved in other primates but not rodents. The RNA transcript was expressed at equal or greater levels in the human striatum compared with the two annotated transcripts, PDE10A1 and PDE10A2. The PDE10A19 transcript was detected in polysomal fractions; western blotting experiments confirmed that the RNA transcript is translated into protein. Immunocytochemistry studies using transfected mouse striatal and cortical neurons demonstrated that the PDE10A19 protein distributes to the cytosol, like PDE10A1, and unlike PDE10A2, which is associated with plasma membranes. Immunoprecipitation and immunocytochemical experiments revealed that the PDE10A19 isoform interacts physically with PDE10A2 and, when expressed at elevated levels, interferes with the plasma membrane localization of PDE10A2. These studies illustrate the complexity of PDE10A gene expression in the human brain and highlight the need to unravel the gene's complex and complete coding capabilities along with its transcriptional and translational regulation to guide the development of therapeutic agents that target the protein for the treatment of neuropsychiatric illness.

  18. DIFFERENTIAL AND AGE-DEPENDENT EXPRESSION OF HYPERPOLARIZATION-ACTIVATED, CYCLIC NUCLEOTIDE-GATED CATION CHANNEL ISOFORMS 1–4 SUGGESTS EVOLVING ROLES IN THE DEVELOPING RAT HIPPOCAMPUS

    PubMed Central

    BENDER, R. A.; BREWSTER, A.; SANTORO, B.; LUDWIG, A.; HOFMANN, F.; BIEL, M.; BARAM, T. Z.

    2011-01-01

    Hyperpolarization-activated cation currents (Ih) are found in several brain regions including thalamus and hippocampus. Important functions of these currents in promoting synchronized network activity and in determining neuronal membrane properties have been progressively recognized, but the molecular underpinnings of these currents are only emerging. Ih currents are generated by hyperpolarization-activated, cyclic nucleotide-gated cation channels (HCNs). These channel proteins are encoded by at least four HCN genes, that govern the kinetic and functional properties of the resulting channels. Because of the potential impact of Ih-mediated coordinated neuronal activity on the maturation of the functional hippocampal network, this study focused on determining the expression of the four members of the HCN gene family throughout postnatal hippocampal development at both the regional and single cell level. The results of these experiments demonstrated that HCNs 1, 2 and 4 are differentially expressed in interneuronal and principal cell populations of the rat hippocampal formation. Expression profiles of each HCN isoform evolve during postnatal development, and patterns observed during early postnatal ages differ significantly from those in mature hippocampus. The onset of HCN expression in interneurons of the hippocampus proper precedes that in the dentate gyrus, suggesting that HCN-mediated pacing activity may be generated in hippocampal interneurons prior to those in the hilus. Taken together, these findings indicate an age-dependent spatiotemporal evolution of specific HCN expression in distinct hippocampal cell populations, and suggest that these channels serve differing and evolving functions in the maturation of coordinated hippocampal activity. PMID:11682156

  19. Mouse white adipocytes and 3T3-L1 cells display an anomalous pattern of carnitine palmitoyltransferase (CPT) I isoform expression during differentiation. Inter-tissue and inter-species expression of CPT I and CPT II enzymes.

    PubMed Central

    Brown, N F; Hill, J K; Esser, V; Kirkland, J L; Corkey, B E; Foster, D W; McGarry, J D

    1997-01-01

    The outer mitochondrial membrane enzyme carnitine palmitoyltransferase I (CPT I) represents the initial and regulated step in the beta-oxidation of fatty acids. It exists in at least two isoforms, denoted L (liver) and M (muscle) types, with very different kinetic properties and sensitivities to malonyl-CoA. Here we have examined the relative expression of the CPT I isoforms in two different models of adipocyte differentiation and in a number of rat tissues. Adipocytes from mice, hamsters and humans were also evaluated. Primary monolayer cultures of undifferentiated rat preadipocytes expressed solely L-CPT I, but significant levels of M-CPT I emerged after only 3 days of differentiation in vitro; in the mature cell M-CPT I predominated. In sharp contrast, the murine 3T3-L1 preadipocyte expressed essentially exclusively L-CPT I, both in the undifferentiated state and throughout the differentiation process in vitro. This was also true of the mature mouse white fat cell. Fully developed adipocytes from the hamster and human behaved similarly to those of the rat. Thus the mouse white fat cell differs fundamentally from those of the other species examined in terms of tis choice of a key regulatory enzyme in fatty acid metabolism. In contrast, brown adipose tissue from all three rodents displayed the same isoform profiles, each expressing overwhelmingly M-CPT I. Northern blot analysis of other rat tissues established L-CPT I as the dominant isoform not only in liver but also in kidney, lung, ovary, spleen, brain, intestine and pancreatic islets. In addition to its primacy in skeletal muscle, heart and fat, M-CPT I was also found to dominate the testis. The same inter-tissue isoform pattern (with the exception of white fat) was found in the mouse. Taken together, the data bring to light an intriguing divergence between white adipocytes of the mouse and other mammalian species. They also raise a cautionary note that should be considered in the choice of animal model used

  20. Crosstalk between Smad2/3 and specific isoforms of ERK in TGF-β1-induced TIMP-3 expression in rat chondrocytes.

    PubMed

    Zhu, Yanhui; Gu, Jianhua; Zhu, Tong; Jin, Chen; Hu, Xiaopeng; Wang, Xiang

    2017-02-23

    This study investigated the roles of ERK1 and ERK2 in transforming growth factor-β1 (TGF-β1)-induced tissue inhibitor of metalloproteinases-3 (TIMP-3) expression in rat chondrocytes, and the specific roles of ERK1 and ERK2 in crosstalk with Smad2/3 were investigated to demonstrate the molecular mechanism of ERK1/2 regulation of TGF-β1 signalling. To examine the interaction of specific isoforms of ERK and the Smad2/3 signalling pathway, chondrocytes were infected with LV expressing either ERK1 or ERK2 siRNA and stimulated with or without TGF-β1. At indicated time-points, TIMP-3 expression was determined by real-time PCR and Western blotting; p-Smad3, nuclear p-Smad3, Smad2/3, p-ERK1/2 and ERK1/2 levels were assessed. And then, aggrecan, type II collagen and the intensity of matrix were examined. TGF-β1-induced TIMP-3 expression was significantly inhibited by ERK1 knock-down, and the decrease in TIMP-3 expression was accompanied by a reduction of p-Smad3 in ERK1 knock-down cells. Knock-down of ERK2 had no effect on neither TGF-β1-induced TIMP-3 expression nor the quantity of p-Smad3. Moreover, aggrecan, type II collagen expression and the intensity of matrix were significantly suppressed by ERK1 knock-down instead of ERK2 knock-down. Taken together, ERK1 and ERK2 have different roles in TGF-β1-induced TIMP-3 expression in rat chondrocytes. ERK1 instead of ERK2 can regulate TGF-β/Smad signalling, which may be the mechanism through which ERK1 regulates TGF-β1-induced TIMP-3 expression.

  1. NF-κB and Androgen Receptor Variant 7 Induce Expression of SRD5A Isoforms and Confer 5ARI Resistance

    PubMed Central

    Austin, David C.; Strand, Douglas W.; Love, Harold L.; Franco, Omar E.; Grabowska, Magdalena M.; Miller, Nicole L.; Hameed, Omar; Clark, Peter E.; Matusik, Robert J.; Jin, Ren J.; Hayward, Simon W.

    2016-01-01

    BACKGROUND Benign prostatic hyperplasia (BPH) is treated with 5α-reductase inhibitors (5ARI). These drugs inhibit the conversion of testosterone to dihydrotestosterone resulting in apoptosis and prostate shrinkage. Most patients initially respond to 5ARIs; however, failure is common especially in inflamed prostates, and often results in surgery. This communication examines a link between activation of NF-κB and increased expression of SRD5A2 as a potential mechanism by which patients fail 5ARI therapy. METHODS Tissue was collected from “Surgical” patients, treated specifically for lower urinary tract symptoms secondary to advanced BPH; and, cancer free transition zone from “Incidental” patients treated for low grade, localized peripheral zone prostate cancer. Clinical, molecular and histopathological profiles were analyzed. Human prostatic stromal and epithelial cell lines were genetically modified to regulate NF-κB activity, androgen receptor (AR) full length (AR-FL), and AR variant 7 (AR-V7) expression. RESULTS SRD5A2 is upregulated in advanced BPH. SRD5A2 was significantly associated with prostate volume determined by Transrectal Ultrasound (TRUS), and with more severe lower urinary tract symptoms (LUTS) determined by American Urological Association Symptom Score (AUASS). Synthesis of androgens was seen in cells in which NF-κB was activated. AR-FL and AR-V7 expression increased SRD5A2 expression while forced activation of NF-κB increased all three SRD5A isoforms. Knockdown of SRD5A2 in the epithelial cells resulted in significant reduction in proliferation, AR target gene expression, and response to testosterone (T). In tissue recombinants, canonical NF-κB activation in prostatic epithelium elevated all three SRD5A isoforms and resulted in in vivo growth under castrated conditions. CONCLUSION Increased BPH severity in patients correlates with SRD5A2 expression. We demonstrate that NF-κB and AR-V7 upregulate SRD5A expression providing a mechanism

  2. Ion channel and lipid scramblase activity associated with expression of TMEM16F/ANO6 isoforms.

    PubMed

    Scudieri, Paolo; Caci, Emanuela; Venturini, Arianna; Sondo, Elvira; Pianigiani, Giulia; Marchetti, Carla; Ravazzolo, Roberto; Pagani, Franco; Galietta, Luis J V

    2015-09-01

    TMEM16F is a membrane protein with possible dual function as an ion channel and a phospholipid scramblase. The properties of ion channels associated with TMEM16F and the link between ion channel and scramblase activity are a matter of debate. We studied the properties of four isoforms of TMEM16F generated by alternative splicing. Upregulation of three TMEM16F isoforms or silencing of endogenous TMEM16F increased and decreased, respectively, both scramblase and channel activities. Introduction of an activating mutation in TMEM16F sequence caused a marked increase in phosphatidylserine scrambling and in ion transport indicating direct involvement of the protein in both functions. TMEM16F, also known as ANO6, is a membrane protein that has been associated with phospholipid scramblase and ion channel activity. However, the characteristics of TMEM16F-dependent channels, particularly the ion selectivity, are a matter of debate. Furthermore, the direct involvement of TMEM16F in phospholipid scrambling has been questioned. We studied the properties of different TMEM16F variants generated by alternative splicing. Using whole-cell patch-clamp recordings, we found that V1, V2 and V5 variants generated membrane currents activated by very high (micromolar) intracellular Ca(2+) concentrations and positive membrane potentials. These variants showed different degrees of Ca(2+) sensitivity and kinetics of activation but similar ion permeability, characterized by a slight selectivity for Cl(-) over Na(+) . A fourth variant (V3) showing a unique carboxy-terminus was devoid of activity, in agreement with its intracellular localization. We also measured scramblase activity using the binding of annexin V to detect phosphatidylserine on the cell surface. V1, V2 and V5 variants were associated with calcium-dependent phosphatidylserine externalization. Interestingly, introduction of an activating mutation, D409G, produced a marked increase in the apparent Ca(2+) sensitivity of TMEM16F

  3. Aestivation Induces Changes in the mRNA Expression Levels and Protein Abundance of Two Isoforms of Urea Transporters in the Gills of the African Lungfish, Protopterus annectens.

    PubMed

    Chng, You R; Ong, Jasmine L Y; Ching, Biyun; Chen, Xiu L; Hiong, Kum C; Wong, Wai P; Chew, Shit F; Lam, Siew H; Ip, Yuen K

    2017-01-01

    The African lungfish, Protopterus annectens, is ammonotelic in water despite being ureogenic. When it aestivates in mucus cocoon on land, ammonia is detoxified to urea. During the maintenance phase of aestivation, urea accumulates in the body, which is subsequently excreted upon arousal. Urea excretion involves urea transporters (UT/Ut). This study aimed to clone and sequence the ut isoforms from the gills of P. annectens, and to test the hypothesis that the mRNA and/or protein expression levels of ut/Ut isoforms could vary in the gills of P. annectens during the induction, maintenance, and arousal phases of aestivation. Two isoforms of ut, ut-a2a and ut-a2b, were obtained from the gills of P. annectens. ut-a2a consisted of 1227 bp and coded for 408 amino acids with an estimated molecular mass of 44.7 kDa, while ut-a2b consisted of 1392 bp and coded for 464 amino acids with an estimated molecular mass of 51.2 kDa. Ut-a2a and Ut-a2b of P. annectens had a closer phylogenetic relationship with Ut/UT of tetrapods than Ut of fishes. While the mRNA expression pattern of ut-a2a and ut-a2b across various tissues of P. annectens differed, the transcript levels of ut-a2a and ut-a2b in the gills were comparable, indicating that they might be equally important for branchial urea excretion during the initial arousal phase of aestivation. During the maintenance phase of aestivation, the transcript level of ut-a2a increased significantly, but the protein abundance of Ut-a2a remained unchanged in the gills of P. annectens. This could be an adaptive feature to prepare for an increase in the production of Ut-a2a upon arousal. Indeed, arousal led to a significant increase in the branchial Ut-a2a protein abundance. Although the transcript level of ut-a2b remained unchanged, there were significant increases in the protein abundance of Ut-a2b in the gills of P. annectens throughout the three phases of aestivation. The increase in the protein abundance of Ut-a2b during the maintenance

  4. Aestivation Induces Changes in the mRNA Expression Levels and Protein Abundance of Two Isoforms of Urea Transporters in the Gills of the African Lungfish, Protopterus annectens

    PubMed Central

    Chng, You R.; Ong, Jasmine L. Y.; Ching, Biyun; Chen, Xiu L.; Hiong, Kum C.; Wong, Wai P.; Chew, Shit F.; Lam, Siew H.; Ip, Yuen K.

    2017-01-01

    The African lungfish, Protopterus annectens, is ammonotelic in water despite being ureogenic. When it aestivates in mucus cocoon on land, ammonia is detoxified to urea. During the maintenance phase of aestivation, urea accumulates in the body, which is subsequently excreted upon arousal. Urea excretion involves urea transporters (UT/Ut). This study aimed to clone and sequence the ut isoforms from the gills of P. annectens, and to test the hypothesis that the mRNA and/or protein expression levels of ut/Ut isoforms could vary in the gills of P. annectens during the induction, maintenance, and arousal phases of aestivation. Two isoforms of ut, ut-a2a and ut-a2b, were obtained from the gills of P. annectens. ut-a2a consisted of 1227 bp and coded for 408 amino acids with an estimated molecular mass of 44.7 kDa, while ut-a2b consisted of 1392 bp and coded for 464 amino acids with an estimated molecular mass of 51.2 kDa. Ut-a2a and Ut-a2b of P. annectens had a closer phylogenetic relationship with Ut/UT of tetrapods than Ut of fishes. While the mRNA expression pattern of ut-a2a and ut-a2b across various tissues of P. annectens differed, the transcript levels of ut-a2a and ut-a2b in the gills were comparable, indicating that they might be equally important for branchial urea excretion during the initial arousal phase of aestivation. During the maintenance phase of aestivation, the transcript level of ut-a2a increased significantly, but the protein abundance of Ut-a2a remained unchanged in the gills of P. annectens. This could be an adaptive feature to prepare for an increase in the production of Ut-a2a upon arousal. Indeed, arousal led to a significant increase in the branchial Ut-a2a protein abundance. Although the transcript level of ut-a2b remained unchanged, there were significant increases in the protein abundance of Ut-a2b in the gills of P. annectens throughout the three phases of aestivation. The increase in the protein abundance of Ut-a2b during the maintenance

  5. Cell-surface expression of PrPC and the presence of scrapie prions in the blood of goats.

    PubMed

    Dassanayake, Rohana P; Schneider, David A; Herrmann-Hoesing, Lynn M; Truscott, Thomas C; Davis, William C; O'Rourke, Katherine I

    2012-05-01

    Although host-encoded prion protein (PrP(C)) expression in ovine PBMCs and prion infectivity in scrapie-infected sheep blood have been demonstrated, such studies have not been reported in goats. Therefore, this study characterized cell-surface expression of PrP(C) on PBMC subsets derived from normal goats and sheep, by flow cytometry, and determined prion infectivity in blood from a scrapie-infected goat using a transfusion bioassay in goat kids. Cell-surface PrP(C) expression was detected on all subsets of goat PBMCs. The highest PrP(C) cell-surface expression was found in CD2(+) T lymphocytes in goats. Transmission of infection was detected in all three recipients who received whole blood from a goat with classical scrapie. It was concluded that caprine PBMCs express PrP(C) similarly to sheep but with relative differences among PBMCs subsets, and that blood-borne infectious prions can be detected in scrapie-infected goats. Thus, similar to sheep, goat blood may be a suitable diagnostic target for the detection of scrapie infection.

  6. Ecdysone receptor isoform-B mediates soluble trehalase expression to regulate growth and development in the mirid bug, Apolygus lucorum (Meyer-Dür).

    PubMed

    Tan, Y-A; Xiao, L-B; Zhao, J; Xiao, Y-F; Sun, Y; Bai, L-X

    2015-12-01

    Ecdysone receptor (EcR) is the hormonal receptor of ecdysteroids and strictly regulates growth and development in insects. However, the action mechanism of EcR is not very clear. In this study, the cDNA of EcR isoform-B was cloned from Apolygus lucorum (AlEcR-B) and its expression profile was investigated. We reduced AlEcR-B mRNA expression using systemic RNA interference in vivo, and obtained knockdown specimens. Examination of these specimens indicated that AlEcR-B is required for nymphal survival, and that reduced expression is associated with longer development time and lower nymphal weight. To investigate the underlying molecular mechanism of the observed suppression effects, we selected trehalase for a detailed study. Transcript encoding soluble trehalase (AlTre-1) was up-regulated by 20-hydroxyecdysone and in agreement with the mRNA expression of AlEcR-B. The expression profile of AlTre-1, soluble trehalase activity and translated protein level in the midgut of surviving nymphs were down-regulated, compared with controls, after the knockdown expression of AlEcR-B. By contrast, membrane-bound trehalase activity, the related gene expression and translated protein level remained at their initial levels. However, trehalose content significantly increased and the glucose content significantly decreased under the same conditions. We propose that AlEcR-B controls normal carbohydrate metabolism by mediating the expression of AlTre-1 to regulate the growth and development in A. lucorum, which provide an extended information into the functions of AlEcR-B.

  7. Regulation of DM-20 mRNA expression and intracellular translocation of glutathione-S-transferase pi isoform during oligodendrocyte differentiation in the adult rat spinal cord.

    PubMed

    Kitada, Masaaki; Takeda, Kazuya; Dezawa, Mari

    2016-07-01

    We previously demonstrated that NG2-positive oligodendrocyte precursor cells (OPCs) do not express DM-20 mRNA and identified a distinct DM-20 mRNA-positive cell population expressing glutathione-S-transferase pi isoform (GST-pi) in the nucleus (GST-pi(Nuc)) of the adult rat spinal cord. As GST-pi intranuclear localization correlates with progenitor cell properties, we examined the differentiation status of this cell population under the intensive 5-bromo-2'-deoxyuridine (BrdU) administration method, consisting of intraperitoneal BrdU injections every 2 h for 48 h. We observed that a certain population of proliferating/proliferated cells expressed DM-20 mRNA, and sometimes two proliferating/proliferated cells were observed still attached to each other. We performed triple staining for BrdU, DM-20 mRNA, and NG2 and found pairs of neighboring BrdU-positive cells, which were considered to originate from the same progenitor cells and where both cells expressed DM-20 mRNA. Triple staining for BrdU, DM-20 mRNA, and GST-pi detected proliferating/proliferated cells exhibiting the GST-pi(Nuc)/DM-20 mRNA-positive expression pattern. These findings suggested the presence of a GST-pi(Nuc)/DM-20 mRNA-positive oligodendrocyte-lineage progenitor cell population in the adult rat spinal cord. However, we did not find any pair of neighboring BrdU-positive cells with this expression pattern. These observations collectively support the idea that GST-pi(Nuc)/DM-20 mRNA-expressing cells are the progeny of NG2-positive OPCs rather than a novel type of oligodendrocyte-lineage progenitor cells and that DM-20 mRNA expression is dynamically regulated during differentiation of OPCs into oligodendrocytes.

  8. Expression of beta 1B integrin isoform in CHO cells results in a dominant negative effect on cell adhesion and motility

    PubMed Central

    1994-01-01

    The integrin subunit beta 1B, a beta 1 isoform with a unique sequence at the cytoplasmic domain, forms heterodimers with integrin alpha chains and binds fibronectin, but it does not localize to focal adhesion sites (Balzac, F., A. Belkin, V. Koteliansky, Y. Balabanow, F. Altruda, L. Silengo, and G. Tarone. 1993. J. Cell Biol. 121:171-178). Here we analyze the functional properties of human beta 1B by expressing it in hamster CHO cells. When stimulated by specific antibodies, beta 1B does not trigger tyrosine phosphorylation of a 125- kD cytosolic protein, an intracellular signalling pathway that is activated both by the endogenous hamster or the transfected human beta 1A. Moreover, expression of beta 1B results in reduced spreading on fibronectin and laminin, but not on vitronectin. Expression of beta 1B also results in severe reduction of cell motility in the Boyden chamber assay. Reduced cell spreading and motility could not be accounted for by preferential association of beta 1B with a given integrin alpha subunit. These data, together with our previous results, indicate that beta 1B interferes with beta 1A function when expressed in CHO cells resulting in a dominant negative effect on cell adhesion and migration. PMID:7523423

  9. Expression of beta 1B integrin isoform in CHO cells results in a dominant negative effect on cell adhesion and motility.

    PubMed

    Balzac, F; Retta, S F; Albini, A; Melchiorri, A; Koteliansky, V E; Geuna, M; Silengo, L; Tarone, G

    1994-10-01

    The integrin subunit beta 1B, a beta 1 isoform with a unique sequence at the cytoplasmic domain, forms heterodimers with integrin alpha chains and binds fibronectin, but it does not localize to focal adhesion sites (Balzac, F., A. Belkin, V. Koteliansky, Y. Balabanow, F. Altruda, L. Silengo, and G. Tarone. 1993. J. Cell Biol. 121:171-178). Here we analyze the functional properties of human beta 1B by expressing it in hamster CHO cells. When stimulated by specific antibodies, beta 1B does not trigger tyrosine phosphorylation of a 125-kD cytosolic protein, an intracellular signalling pathway that is activated both by the endogenous hamster or the transfected human beta 1A. Moreover, expression of beta 1B results in reduced spreading on fibronectin and laminin, but not on vitronectin. Expression of beta 1B also results in severe reduction of cell motility in the Boyden chamber assay. Reduced cell spreading and motility could not be accounted for by preferential association of beta 1B with a given integrin alpha subunit. These data, together with our previous results, indicate that beta 1B interferes with beta 1A function when expressed in CHO cells resulting in a dominant negative effect on cell adhesion and migration.

  10. Regulation of spblimp1/krox1a, an alternatively transcribed isoform expressed in midgut and hindgut of the sea urchin gastrula.

    PubMed

    Livi, Carolina B; Davidson, Eric H

    2007-01-01

    The sea urchin regulatory gene Spblimp1/krox produces alternatively transcribed and spliced isoforms, 1a and 1b, which have different temporal and spatial patterns of expression. Here we describe a cis-regulatory module that controls the expression of the 1a splice form in the midgut and hindgut at the beginning of gastrulation. Conserved sequence patches revealed by a comparison of the blimp1/krox locus in Strongylocentrotus purpuratus and Lytechinus variegatus genomes were tested by gene transfer, in association with GFP or CAT reporter genes. An expression construct containing a conserved sequence patch immediately 5' of exon 1a included the transcription initiation site for blimp1/krox1a. This construct displays specific mid and hindgut expression, indicating that these are the locations of endogenous blimp1/krox1a transcription during the gastrula stage. Its sequence contains binding sites for Brn1/2/4, Otx, and Blimp1/Krox itself, as predicted in a prior regulatory network analysis.

  11. Histamine H3-receptor isoforms.

    PubMed

    Bakker, R A

    2004-10-01

    Increasing evidence supports a role for HA as a neurotransmitter and neuromodulator in various brain functions, including emotion, cognition, and feeding. The recent cloning of the histamine H3 receptor allowed for the subsequent cloning of a variety of H3 receptor isoforms from different species as well as the H4 receptor. As a result a wide variety of H3-receptor isoforms are now known that display differential brain expression patterns and signalling properties. These recent discoveries are discussed in view of the growing interest of the H3 receptor as a target for the development of potential therapeutics.

  12. Molecular cloning and expression of prostaglandin F2α receptor isoforms during ovulation in the ovarian follicles of Xenopus laevis.

    PubMed

    Liu, Zhiming; Su, Xiurong; Li, Taiwu; Pan, Daodong; Sena, Johnny; Dhillon, Jasvinder

    2010-11-01

    Prostaglandins F2α levels increase during ovulatory period in Xenopus laevis in response to stimulation by gonadotropins and progesterone. PGF2α exerts its effects on ovulation through interaction with its receptor (FP) in ovaries. Little is known about the characteristics of the FP receptor and its regulation during the ovulatory period in non-mammalian species. In the present study, two isoforms of prostaglandin F receptor (FP A and B) cDNAs were isolated from Xenopus laevis ovarian tissues using reverse transcription-polymerase chain reaction (RT-PCR) followed by rapid amplification of cDNA ends (RACE). The cDNAs of FP A and FP B were sequenced. In Xenopus laevis ovary, FP A and B mRNA levels were up-regulated during gonadotropin- and progresterone-induced ovulation in vitro. The mRNA level of FP B was higher than that of FP A. Moreover, FP A and FP B mRNA levels were measured in various tissues including eye, liver, lungs, heart, muscle, ovary, and skin. Overall, FP B mRNA level was approximately 10- to 100-fold higher than that of FP A, except in the muscle and skin where FP A mRNA level was comparable to that of FP B. The results suggest that in Xenopus ovarian follicles FP receptors play an important role during gonadotropin- and progesterone-induced ovulation.

  13. Enhanced expression of metallothionein isoform 3 protein in tumor heterotransplants derived from As+3- and Cd+2-transformed human urothelial cells.

    PubMed

    Zhou, Xu Dong; Sens, Mary Ann; Garrett, Scott H; Somji, Seema; Park, Seongmi; Gurel, Volkan; Sens, Donald A

    2006-10-01

    This laboratory has proposed that the third isoform of the metallothionein gene family (MT-3) might be a biomarker for the development of human bladder cancer. Immunohistochemical staining of MT-3 on archival diagnostic specimens showed that only 2 of 63 (3.17%) benign bladder specimens had even weak reactivity for the MT-3 protein. In contrast, 103 of 107 (96.26%) high-grade urothelial cancers and 17 of 17 (100%) specimens of carcinoma in situ stained positive for the MT-3 protein. For low-grade bladder cancer it was shown that 30 of 48 specimens (62.5%) expressed the MT-3 protein. Using a cell culture model (UROtsa), it was demonstrated that expression of the MT-3 protein was not required for malignant transformation of urothelial cells by either Cd(+2) or As(+3). In contrast, it was shown that the cells transformed by Cd(+2) and As(+3) that did not express the MT-3 gene in cell culture, gained expression of MT-3 when grown as heterotransplants in nude mice. The gain in MT-3 expression when cells were grown as heterotransplants was also shown to occur for the MCF-7, T-47D, Hs 578t, MDA-MB-231 breast cancer, and the PC-3 prostate cancer cell lines. An analysis of MT-3 mRNA and protein expression suggested that a posttranscriptional mechanism was responsible for accumulation of the MT-3 protein. The results provide strong evidence that MT-3 could be a biomarker for the development of high-grade bladder cancer and that the expression of the MT-3 gene is not involved in the in vitro malignant transformation of UROtsa cells by Cd(+2) and As(+3).

  14. Isoform-Level Gene Expression Profiles of Human Y Chromosome Azoospermia Factor Genes and Their X Chromosome Paralogs in the Testicular Tissue of Non-Obstructive Azoospermia Patients.

    PubMed

    Ahmadi Rastegar, Diba; Sharifi Tabar, Mehdi; Alikhani, Mehdi; Parsamatin, Pouria; Sahraneshin Samani, Fazel; Sabbaghian, Marjan; Sadighi Gilani, Mohammad Ali; Mohammad Ahadi, Ali; Mohseni Meybodi, Anahita; Piryaei, Abbas; Ansari-Pour, Naser; Gourabi, Hamid; Baharvand, Hossein; Salekdeh, Ghasem Hosseini

    2015-09-04

    The human Y chromosome has an inevitable role in male fertility because it contains many genes critical for spermatogenesis and the development of the male gonads. Any genetic variation or epigenetic modification affecting the expression pattern of Y chromosome genes may thus lead to male infertility. In this study, we performed isoform-level gene expression profiling of Y chromosome genes within the azoospermia factor (AZF) regions, their X chromosome counterparts, and few autosomal paralogues in testicular biopsies of 12 men with preserved spermatogenesis and 68 men with nonobstructive azoospermia (NOA) (40 Sertoli-cell-only syndrome (SCOS) and 28 premiotic maturation arrest (MA)). This was undertaken using quantitative real-time PCR (qPCR) at the transcript level and Western blotting (WB) and immunohistochemistry (IHC) at the protein level. We profiled the expression of 41 alternative transcripts encoded by 14 AZFa, AZFb, and AZFc region genes (USP9Y, DDX3Y, XKRY, HSFY1, CYORF15A, CYORF15B, KDM5D, EIF1AY, RPS4Y2, RBMY1A1, PRY, BPY2, DAZ1, and CDY1) as well as their X chromosome homologue transcripts and a few autosomal homologues. Of the 41 transcripts, 18 were significantly down-regulated in men with NOA when compared with those of men with complete spermatogenesis. In contrast, the expression of five transcripts increased significantly in NOA patients. Furthermore, to confirm the qPCR results at the protein level, we performed immunoblotting and IHC experiments (based on 24 commercial and homemade antibodies) that detected 10 AZF-encoded proteins. In addition, their localization in testis cell types and organelles was determined. Interestingly, the two missing proteins, XKRY and CYORF15A, were detected for the first time. Finally, we focused on the expression patterns of the significantly altered genes in 12 MA patients with successful sperm retrieval compared to those of 12 MA patients with failed sperm retrieval to predict the success of sperm retrieval in

  15. Expression of distinct classes of titin isoforms in striated and smooth muscles by alternative splicing, and their conserved interaction with filamins.

    PubMed

    Labeit, Siegfried; Lahmers, Sunshine; Burkart, Christoph; Fong, Chi; McNabb, Mark; Witt, Stephanie; Witt, Christian; Labeit, Dietmar; Granzier, Henk

    2006-09-29

    While the role of titin as a sarcomeric protein is well established, its potential functional role(s) in smooth muscles and non-muscle tissues are controversial. We used a titin exon array to search for which part(s) of the human titin transcriptional unit encompassing 363 exons is(are) expressed in non-striated muscle tissues. Expression profiling of adult smooth muscle tissues (aorta, bladder, carotid, stomach) identified alternatively spliced titin isoforms, encompassing 80 to about 100 exons. These exons code for parts of the titin Z-disk, I-band and A-band regions, allowing the truncated smooth muscle titin isoform to link Z-disks/dense bodies together with thick filaments. Consistent with the array data, Western blot studies detected the expression of approximately 1 MDa smooth muscle titin in adult smooth muscles, reacting with selected Z-disc, I-band, and A-band titin antibodies. Immunofluorescence with these antibodies located smooth muscle titin in the cytoplasm of cultured human aortic smooth muscle cells and in the tunica media of intact adult bovine aorta. Real time PCR studies suggested that smooth muscle titins are expressed from a promoter located 35 kb or more upstream of the transcription initiation site used for striated muscle titin, driving expression of a bi-cistronic mRNA, coding 5' for the anonymous gene FL39502, followed 3' by titin, respectively. Our work showed that smooth muscle and striated muscle titins share in their conserved amino-terminal regions binding sites for alpha-actinin and filamins: Yeast two-hybrid screens using Z2-Zis1 titin baits identified prey clones coding for alpha-actinin-1 and filamin-A from smooth muscle, and alpha-actinin-2/3, filamin-C, and nebulin from skeletal muscle cDNA libraries, respectively. This suggests that the titin Z2-Zis1 domain can link filamins and alpha-actinin together in the periphery of the Z-line/dense bodies in a fashion that is conserved in smooth and striated muscles.

  16. Calmodulin Gene Family in Potato: Developmental and Touch-Induced Expression of the mRNA Encoding a Novel Isoform

    NASA Technical Reports Server (NTRS)

    Takezawa, D.; Liu, Z. H.; An, G.; Poovaiah, B. W.

    1995-01-01

    Eight genomic clones of potato calmodulin (PCM1 to 8) were isolated and characterized. Sequence comparisons of different genes revealed that the deduced amino acid sequence of PCM1 had several unique substitutions, especially in the fourth Ca(2+)-binding area. The expression patterns of different genes were studied by northern analysis using the 3'-untranslated regions as probes. The expression of PCM1, 5, and 8 was highest in the stolon tip and it decreased during tuber development. The expression of PCM6 did not vary much in the tissues tested, except in the leaves, where the expression was lower; whereas, the expression of PCM4 was very low in all the tissues. The expression of PCM2 and PCM3 was not detected in any of the tissues tested. Among these genes, only PCM1 showed increased expression following touch stimulation. To study the regulation of PCM1, transgenic potato plants carrying the PCM1 promoter fused to the beta-glucuronidase (GUS) reporter gene were produced. GUS expression was found to be developmentally regulated and touch-responsive, indicating a positive correlation between the expression of PCM1 and GUS mRNAs. These results suggest that the 5'-flanking region of PCM1 controls developmental and touch-induced expression. X-Gluc staining patterns revealed that GUS localization is high in meristematic tissues such as the stem apex, stolon tip, and vascular regions.

  17. Tissue-specific expression of betaKlotho and fibroblast growth factor (FGF) receptor isoforms determines metabolic activity of FGF19 and FGF21.

    PubMed

    Kurosu, Hiroshi; Choi, Mihwa; Ogawa, Yasushi; Dickson, Addie S; Goetz, Regina; Eliseenkova, Anna V; Mohammadi, Moosa; Rosenblatt, Kevin P; Kliewer, Steven A; Kuro-o, Makoto

    2007-09-14

    The fibroblast growth factor (FGF) 19 subfamily of ligands, FGF19, FGF21, and FGF23, function as hormones that regulate bile acid, fatty acid, glucose, and phosphate metabolism in target organs through activating FGF receptors (FGFR1-4). We demonstrated that Klotho and betaKlotho, homologous single-pass transmembrane proteins that bind to FGFRs, are required for metabolic activity of FGF23 and FGF21, respectively. Here we show that, like FGF21, FGF19 also requires betaKlotho. Both FGF19 and FGF21 can signal through FGFR1-3 bound by betaKlotho and increase glucose uptake in adipocytes expressing FGFR1. Additionally, both FGF19 and FGF21 bind to the betaKlotho-FGFR4 complex; however, only FGF19 signals efficiently through FGFR4. Accordingly, FGF19, but not FGF21, activates FGF signaling in hepatocytes that primarily express FGFR4 and reduces transcription of CYP7A1 that encodes the rate-limiting enzyme for bile acid synthesis. We conclude that the expression of betaKlotho, in combination with particular FGFR isoforms, determines the tissue-specific metabolic activities of FGF19 and FGF21.

  18. Influence of Botulinumtoxin A on the Expression of Adult MyHC Isoforms in the Masticatory Muscles in Dystrophin-Deficient Mice (Mdx-Mice)

    PubMed Central

    Todorov, Teodor

    2016-01-01

    The most widespread animal model to investigate Duchenne muscular dystrophy is the mdx-mouse. In contrast to humans, phases of muscle degeneration are replaced by regeneration processes; hence there is only a restricted time slot for research. The aim of the study was to investigate if an intramuscular injection of BTX-A is able to break down muscle regeneration and has direct implications on the gene expression of myosin heavy chains in the corresponding treated and untreated muscles. Therefore, paralysis of the right masseter muscle was induced in adult healthy and dystrophic mice by a specific intramuscular injection of BTX-A. After 21 days the mRNA expression and protein content of MyHC isoforms of the right and left masseter, temporal, and the tongue muscle were determined using quantitative RT-PCR and Western blot technique. MyHC-IIa and MyHC-I-mRNA expression significantly increased in the paralyzed masseter muscle of control-mice, whereas MyHC-IIb and MyHC-IIx/d-mRNA were decreased. In dystrophic muscles no effect of BTX-A could be detected at the level of MyHC. This study suggests that BTX-A injection is a suitable method to simulate DMD-pathogenesis in healthy mice but further investigations are necessary to fully analyse the BTX-A effect and to generate sustained muscular atrophy in mdx-mice. PMID:27689088

  19. Expression of a novel isoform of Na+/H+ exchanger 3 in the kidney and intestine of banded houndshark, Triakis scyllium

    PubMed Central

    Li, Shanshan; Takabe, Souichirou; Chen, An-Ping; Romero, Michael F.; Umezawa, Takahiro; Nakada, Tsutomu; Hyodo, Susumu; Hirose, Shigehisa

    2013-01-01

    Na+/H+ exchanger 3 (NHE3) provides one of the major Na+ absorptive pathways of the intestine and kidney in mammals, and recent studies of aquatic vertebrates (teleosts and elasmobranchs) have demonstrated that NHE3 is expressed in the gill and plays important roles in ion and acid-base regulation. To understand the role of NHE3 in elasmobranch osmoregulatory organs, we analyzed renal and intestinal expressions and localizations of NHE3 in a marine elasmobranch, Japanese banded houndshark (Triakis scyllium). mRNA for Triakis NHE3 was most highly expressed in the gill, kidney, spiral intestine, and rectum. The kidney and intestine expressed a transcriptional isoform of NHE3 (NHE3k/i), which has a different amino terminus compared with that of NHE3 isolated from the gill (NHE3g), suggesting that NHE3k/i and NHE3g arise from a single gene by alternative promoter usage. Immunohistochemical analyses of the Triakis kidney demonstrated that NHE3k/i is expressed in the apical membrane of a part of the proximal and late distal tubules in the sinus zone. In the bundle zone of the kidney, NHE3k/i was expressed in the apical membrane of the early distal tubules known as the diluting segment. In the spiral intestine and rectum, NHE3k/i was localized toward the apical membrane of the epithelial cells. The transcriptional levels of NHE3k/i were increased in the kidney when Triakis was acclimated in 130% seawater, whereas those in the spiral intestine were increased in fish acclimated in diluted seawater. These results suggest that NHE3 is involved in renal Na+ reabsorption, urine acidification, and intestinal Na+ absorption in elasmobranchs. PMID:23485868

  20. Expression of a novel isoform of Na(+)/H(+) exchanger 3 in the kidney and intestine of banded houndshark, Triakis scyllium.

    PubMed

    Li, Shanshan; Kato, Akira; Takabe, Souichirou; Chen, An-Ping; Romero, Michael F; Umezawa, Takahiro; Nakada, Tsutomu; Hyodo, Susumu; Hirose, Shigehisa

    2013-05-15

    Na(+)/H(+) exchanger 3 (NHE3) provides one of the major Na(+) absorptive pathways of the intestine and kidney in mammals, and recent studies of aquatic vertebrates (teleosts and elasmobranchs) have demonstrated that NHE3 is expressed in the gill and plays important roles in ion and acid-base regulation. To understand the role of NHE3 in elasmobranch osmoregulatory organs, we analyzed renal and intestinal expressions and localizations of NHE3 in a marine elasmobranch, Japanese banded houndshark (Triakis scyllium). mRNA for Triakis NHE3 was most highly expressed in the gill, kidney, spiral intestine, and rectum. The kidney and intestine expressed a transcriptional isoform of NHE3 (NHE3k/i), which has a different amino terminus compared with that of NHE3 isolated from the gill (NHE3g), suggesting that NHE3k/i and NHE3g arise from a single gene by alternative promoter usage. Immunohistochemical analyses of the Triakis kidney demonstrated that NHE3k/i is expressed in the apical membrane of a part of the proximal and late distal tubules in the sinus zone. In the bundle zone of the kidney, NHE3k/i was expressed in the apical membrane of the early distal tubules known as the diluting segment. In the spiral intestine and rectum, NHE3k/i was localized toward the apical membrane of the epithelial cells. The transcriptional levels of NHE3k/i were increased in the kidney when Triakis was acclimated in 130% seawater, whereas those in the spiral intestine were increased in fish acclimated in diluted seawater. These results suggest that NHE3 is involved in renal Na(+) reabsorption, urine acidification, and intestinal Na(+) absorption in elasmobranchs.

  1. Identification and sexually dimorphic expression of vasa isoforms in Dabry's sturgeon (Acipenser dabryanus), and functional analysis of vasa 3'-untranslated region.

    PubMed

    Ye, Huan; Yue, Hua-Mei; Yang, Xiao-Ge; Li, Chuang-Ju; Wei, Qi-Wei

    2016-10-01

    Germ cells are set aside from somatic cells early in embryogenesis, and are responsible for transmitting genetic information through generations. Vasa is a highly conserved germ cell marker across animal phyla, and widely used to label primordial germ cells. Dabry's sturgeon is a rare and endangered species distributed solely in the Yangtze River basin. Here, seven vasa isoforms, named Advasa1-7, were isolated and characterized in Dabry's sturgeon. RT-PCR and western blot analyses revealed that vasa mRNA and protein were mainly restricted to the testis and ovary, but exhibited sexually dimorphic expression. Cellular and subcellular localization uncovered that Advasa mRNA and protein displayed mitotic and meiotic expression in females, and mainly showed mitotic expression in males; surprisingly, they exhibited both cytoplasmic and nuclear expression in the ovarian germ cells, while showing exclusively cytoplasmic expression in the testicular germ cells. By microinjecting chimeric RNA consisting of the red fluorescent protein coding region and the Advasa 3'-untranslated region into embryos of Dabry's sturgeon, zebrafish and medaka, we demonstrated that it had the ability to visualize primordial germ cells (PGCs) in Dabry's sturgeon and zebrafish but not in medaka. It seemed that the machinery of vasa 3'UTR RNA localization was conserved between Dabry's sturgeon and ostariophysan, while possibly changed during the divergence of euteleosts and ostariophysan. Finally, Dabry's sturgeon PGCs moved on the yolk ball, and migrated toward the genital ridge via mesenchyme. Taken together, these results provide new information for vasa expression pattern and function, and lay a foundation for PGC cryopreservation and conservation of Dabry's sturgeon.

  2. Basal and metal-induced expression of metallothionein isoform 1 and 2 genes in the RWPE-1 human prostate epithelial cell line.

    PubMed

    Albrecht, Amy L; Singh, Rajendra K; Somji, Seema; Sens, Mary Ann; Sens, Donald A; Garrett, Scott H

    2008-04-01

    The human prostate gland has low basal expression of the metallothionein-1 and -2 proteins. In prostate cancer, MT-1/2 protein expression is variable and correlates directly with the increasing Gleason score of the tumor. The goal of the present study was to determine if the RWPE-1 cell line is a good model to elucidate the mechanisms underlying the alterations in MT-1/2 expression that occur during the development of prostate cancer. It was shown that the RWPE-1 cell line and in situ prostate tissue have identical expression profiles of MT-1 and MT-2 isoform-specific mRNAs (MT-1E, MT-1X and MT-2A) and similar levels of MT-1/2 protein. It was also shown that the RWPE-1 cells respond to Zn(+2) and Cd(+2) exposure by induction of the basally expressed MT mRNAs and the accumulation of high levels MT-1/2 protein (in excess of 10% of total protein). It was also shown that additional MT-1 mRNAs were expressed when the cells were exposed to either metal; MT-1A, MT-1F, MT-G and MT-1H for Cd(+2)-exposed cells; and, MT-1F, MT-G and MT-1H for Zn(+2)-exposed cells. The results suggest that RWPE-1 cells may be a valuable system to define the interplay between Zn(+2) concentration, Cd(+2) exposure and MT in normal and malignant prostate epithelial cells.

  3. Cloning of a novel phospholipase C-delta isoform from pacific purple sea urchin (Strongylocentrotus purpuratus) gametes and its expression during early embryonic development.

    PubMed

    Coward, Kevin; Owen, Helen; Poustka, Albert J; Hibbitt, Olivia; Tunwell, Richard; Kubota, Hiroki; Swann, Karl; Parrington, John

    2004-01-23

    Calcium (Ca(2+)) is a ubiquitous intracellular messenger, controlling a diverse range of cellular processes, including fertilization and development of the embryo. One of the key mechanisms involved in triggering intracellular calcium release is the generation of the second messenger inositol-1,4,5-phosphate (IP(3)) by the phospholipase C (PLC) class of enzymes. Although five distinct forms of PLC have been identified in mammals (beta, gamma, delta, epsilon, and zeta), only one, PLCgamma, has thus far been detected in echinoderms. In the present study, we describe the isolation of a cDNA encoding a novel PLC isoform of the delta (delta) subclass, PLC-deltasu, from the egg of the Pacific purple sea urchin Strongylocentrotus purpuratus. We also demonstrate the presence of this PLC within the sperm and in the early embryo. The PLC-deltasu cDNA (2.44kb) encodes a 742 amino acid polypeptide with an open reading frame of 84.6kDa and a pI of 6.04. All of the characteristic domains found in mammalian PLCdelta isoforms (PH domain, EF hands, an X-Y catalytic region, and a C2 domain) are present in PLC-deltasu. A homology search revealed that PLC-deltasu shares most sequence identity with bovine PLCdelta2 (39%). We present evidence that PLC-deltasu is expressed in unfertilized eggs, fertilized eggs, and in the early embryo. In addition to Northern and polymerase chain reaction (PCR) analyses, in situ hybridization experiments further demonstrated that the embryonic regions within which the PLC-deltasu transcript can be detected during early embryonic development are associated with the highest levels of proliferative activity, suggesting a possible involvement with metabolism or cell cycle regulation.

  4. Characterization, cloning, and heterologous expression of a subtilisin-like serine protease gene VlPr1 from Verticillium lecanii.

    PubMed

    Yu, Gang; Liu, Jin-Liang; Xie, Li-Qin; Wang, Xue-Liang; Zhang, Shi-Hong; Pan, Hong-Yu

    2012-12-01

    The entomopathogenic fungus Verticillium lecanii is a well-known biocontrol agent. V. lecanii produces subtilisin-like serine protease (Pr1), which is important in the biological control activity of some insect pests by degrading insect cuticles. In this study, a subtilisin-like serine protease gene VlPr1 was cloned from the fungus and the VlPr1 protein was expressed in Escherichia coli. The VlPr1 gene contains an open reading frame (ORF) interrupted by three short introns, and encodes a protein of 379 amino acids. Protein sequence analysis revealed high homology with subtilisin serine proteases. The molecular mass of the protease was 38 kDa, and the serine protease exhibited its maximal activity at 40°C and pH 9.0. Protease activity was also affected by Mg(2+) and Ca(2+) concentration. The protease showed inhibitory activity against several plant pathogens, especially towards Fusarium moniliforme.

  5. Effect of Exercise Intensity on Isoform-Specific Expressions of NT-PGC-1 α mRNA in Mouse Skeletal Muscle

    PubMed Central

    Wen, Xingyuan; Wu, Jing; Wang, Jianzhu; Zhang, Yaliang; Gettys, Thomas W.

    2014-01-01

    PGC-1α is an inducible transcriptional coactivator that regulates mitochondrial biogenesis and cellular energy metabolism in skeletal muscle. Recent studies have identified two additional PGC-1α transcripts that are derived from an alternative exon 1 (exon 1b) and induced by exercise. Given that the PGC-1α gene also produces NT-PGC-1α transcript by alternative 3′ splicing between exon 6 and exon 7, we have investigated isoform-specific expression of NT-PGC-1α mRNA in mouse skeletal muscle during physical exercise with different intensities. We report here that NT-PGC-1α-a mRNA expression derived from a canonical exon 1 (exon 1a) is increased by high-intensity exercise and AMPK activator AICAR in mouse skeletal muscle but not altered by low- and medium-intensity exercise and β2-adrenergic receptor agonist clenbuterol. In contrast, the alternative exon 1b-driven NT-PGC-1α-b (PGC-1α4) and NT-PGC-1α-c are highly induced by low-, medium-, and high-intensity exercise, AICAR, and clenbuterol. Ectopic expression of NT-PGC-1α-a in C2C12 myotube cells upregulates myosin heavy chain (MHC I, MHC II a) and Glut4, which represent oxidative fibers, and promotes the expression of mitochondrial genes (Cyc1, COX5B, and ATP5B). In line with gene expression data, citrate synthase activity was significantly increased by NT-PGC-1α-a in C2C12 myotube cells. Our results indicate the regulatory role for NT-PGC-1α-a in mitochondrial biogenesis and adaptation of skeletal muscle to endurance exercise. PMID:25136584

  6. CCAAT/enhancer binding protein β negatively regulates progesterone receptor expression in human glioblastoma cells.

    PubMed

    Hansberg-Pastor, Valeria; González-Arenas, Aliesha; Camacho-Arroyo, Ignacio

    2017-01-05

    Many progesterone (P4) actions are mediated by its intracellular receptor (PR), which has two isoforms (PR-A and PR-B) differentially transcribed from separate promoters of a single gene. In glioblastomas, the most frequent and aggressive brain tumors, PR-B is the predominant isoform. In an in silico analysis we showed putative CCAAT/Enhancer Binding Protein (C/EBP) binding sites at PR-B promoter. We evaluated the role of C/EBPβ in PR-B expression regulation in glioblastoma cell lines, which expressed different ratios of PR and C/EBPβ isoforms (LAP1, LAP2, and LIP). ChIP assays showed a significant basal binding of C/EBPβ, specific protein 1 (Sp1) and estrogen receptor alpha (ERα) to PR-B promoter. C/EBPβ knockdown increased PR-B expression and treatment with estradiol (E2) reduced C/EBPβ binding to the promoter and up-regulated PR-B expression. P4 induced genes were differently regulated when CEBP/β was silenced. These data show that C/EBPβ negatively regulates PR-B expression in glioblastoma cells.

  7. Insulin Restores Gestational Diabetes Mellitus–Reduced Adenosine Transport Involving Differential Expression of Insulin Receptor Isoforms in Human Umbilical Vein Endothelium

    PubMed Central

    Westermeier, Francisco; Salomón, Carlos; González, Marcelo; Puebla, Carlos; Guzmán-Gutiérrez, Enrique; Cifuentes, Fredi; Leiva, Andrea; Casanello, Paola; Sobrevia, Luis

    2011-01-01

    OBJECTIVE To determine whether insulin reverses gestational diabetes mellitus (GDM)–reduced expression and activity of human equilibrative nucleoside transporters 1 (hENT1) in human umbilical vein endothelium cells (HUVECs). RESEARCH DESIGN AND METHODS Primary cultured HUVECs from full-term normal (n = 44) and diet-treated GDM (n = 44) pregnancies were used. Insulin effect was assayed on hENT1 expression (protein, mRNA, SLC29A1 promoter activity) and activity (initial rates of adenosine transport) as well as endothelial nitric oxide (NO) synthase activity (serine1177 phosphorylation, l-citrulline formation). Adenosine concentration in culture medium and umbilical vein blood (high-performance liquid chromatography) as well as insulin receptor A and B expression (quantitative PCR) were determined. Reactivity of umbilical vein rings to adenosine and insulin was assayed by wire myography. Experiments were in the absence or presence of l-NG-nitro-l-arginine methyl ester (l-NAME; NO synthase inhibitor) or ZM-241385 (an A2A-adenosine receptor antagonist). RESULTS Umbilical vein blood adenosine concentration was higher, and the adenosine- and insulin-induced NO/endothelium-dependent umbilical vein relaxation was lower in GDM. Cells from GDM exhibited increased insulin receptor A isoform expression in addition to the reported NO–dependent inhibition of hENT1-adenosine transport and SLC29A1 reporter repression, and increased extracellular concentration of adenosine and NO synthase activity. Insulin reversed all these parameters to values in normal pregnancies, an effect blocked by ZM-241385 and l-NAME. CONCLUSIONS GDM and normal pregnancy HUVEC phenotypes are differentially responsive to insulin, a phenomenon where insulin acts as protecting factor for endothelial dysfunction characteristic of this syndrome. Abnormal adenosine plasma levels, and potentially A2A-adenosine receptors and insulin receptor A, will play crucial roles in this phenomenon in GDM. PMID:21515851

  8. Induction of nitrate uptake in maize roots: expression of a putative high-affinity nitrate transporter and plasma membrane H+-ATPase isoforms.

    PubMed

    Santi, Simonetta; Locci, Geraldine; Monte, Rossella; Pinton, Roberto; Varanini, Zeno

    2003-08-01

    An investigation was carried out to assess the effect of nitrate supply on the root plasma membrane (PM) H+-ATPase of etiolated maize (Zea mays L.) seedlings grown in hydroponics. The treatment induced higher uptake rates of the anion and the expression of a putative high-affinity nitrate transporter gene (ZmNRT2.1), the first to be identified in maize. Root PM H+-ATPase activity displayed a similar time-course pattern as that of net nitrate uptake and investigations were carried out to determine which of the two isoforms reported to date in maize, MHA1 and 2, responded to the treatment. MHA1 was not expressed under the conditions analysed. Genome analysis revealed that MHA2, described as the most abundant form in all maize tissues, was not present in the maize hybrid investigated, but a similar form was found instead and named MHA3. A second gene (named MHA4) was also identified and partially sequenced. Both genes, classified as members of the PM H+-ATPase subfamily II, responded to nitrate supply, although to different degrees: MHA4, in particular, proved more sensitive than MHA3, with a greater up- and down-regulation in response to the treatment. Increased expression of subfamily II genes resulted in higher steady-state levels of the enzyme in the root tissues and enhanced ATP-hydrolysing activity. The results support the idea that greater proton-pumping activity is required when nitrate inflow increases and suggest that nitrate may be the signal triggering the expression of the two members of PM H+-ATPase subfamily II.

  9. Proline residues in transmembrane segment IV are critical for activity, expression and targeting of the Na+/H+ exchanger isoform 1.

    PubMed Central

    Slepkov, Emily R; Chow, Signy; Lemieux, M Joanne; Fliegel, Larry

    2004-01-01

    NHE1 (Na+/H+ exchanger isoform 1) is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammalian cells. Proline residues within transmembrane segments have unusual properties, acting as helix breakers and increasing flexibility of membrane segments, since they lack an amide hydrogen. We examined the importance of three conserved proline residues in TM IV (transmembrane segment IV) of NHE1. Pro167 and Pro168 were mutated to Gly, Ala or Cys, and Pro178 was mutated to Ala. Pro168 and Pro178 mutant proteins were expressed at levels similar to wild-type NHE1 and were targeted to the plasma membrane. However, the mutants P167G (Pro167-->Gly), P167A and P167C were expressed at lower levels compared with wild-type NHE1, and a significant portion of P167G and P167C were retained intracellularly, possibly indicating induced changes in the structure of TM IV. P167G, P167C, P168A and P168C mutations abolished NHE activity, and P167A and P168G mutations caused markedly decreased activity. In contrast, the activity of the P178A mutant was not significantly different from that of wild-type NHE1. The results indicate that both Pro167 and Pro168 in TM IV of NHE1 are required for normal NHE activity. In addition, mutation of Pro167 affects the expression and membrane targeting of the exchanger. Thus both Pro167 and Pro168 are strictly required for NHE function and may play critical roles in the structure of TM IV of the NHE. PMID:14680478

  10. Differential expression of the L- and S-isoforms of myelin associated glycoprotein (MAG) in oligodendrocyte unit phenotypes in the adult rat anterior medullary velum.

    PubMed

    Butt, A M; Ibrahim, M; Gregson, N; Berry, M

    1998-04-01

    We have previously demonstrated differences in the expression of carbonic anhydrase II (CAII) in oligodendrocyte units myelinating small and large diameter fibres in the anterior medullary velum (AMV) of the adult rat (each unit comprises the cell body, processes and myelin sheaths). Others have indicated that myelin composition may also vary with respect to myelin basic protein (MBP) and proteolipid protein (PLP), and the small (S)- and large (L)-isoforms of myelin associated glycoprotein (MAG). In this study, we have determined the expression of myelin proteins in oligodendrocyte unit phenotypes I-IV, which myelinate fibres ranging in diameter from 0.3-12 microns diameter in the AMV, by using double immunolabelling for Rip, which labels entire units, and MBP, PLP, myelin oligodendrocyte glycoprotein (MOG), L-MAG and S-MAG. We show differences in the expression of L- and S-MAG in units which myelinate different diameter fibres: (1) type I/II units myelinating small diameter fibres had a L-MAG+/S-MAG-/CAII+ phenotype; (2) type II/III units myelinating different diameter fibres had a L-MAG+/S-MAG+/CAII+ phenotype; (3) type III/IV units myelinated large diameter fibres had a L-MAG+/S-MAG+/CAII- phenotype. All units, irrespective of fibre diameter, expressed Rip, MBP, PLP and MOG. The results indicate that type I-IV units may be variants of a single oligodendrocyte population and that phenotypic differences are determined by the diameter of fibres within the unit. The possible significance of metabolic and biochemical differences between oligodendrocytes myelinating small and large diameter axons are discussed with reference to the pathology of demyelination.

  11. Delayed Parturition and Altered Myometrial Progesterone Receptor Isoform A Expression in Mice Null for Kruppel-like Factor 9

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pre-term and delayed labor conditions are devastating health problems, with currently unknown etiologies. We previously showed that the transcription factor Krüppel-like factor 9 (KLF9) influences the expression and/or transcriptional activity of receptors for estrogen and progesterone in endometria...

  12. Selective expression of the type 3 isoform of ryanodine receptor Ca{sup 2+} release channel (RyR3) in a subset of slow fibers in diaphragm and cephalic muscles of adult rabbits

    SciTech Connect

    Conti, Antonio; Reggiani, Carlo; Sorrentino, Vincenzo . E-mail: v.sorrentino@unisi.it

    2005-11-11

    The expression pattern of the RyR3 isoform of Ca{sup 2+} release channels was analysed by Western blot in neonatal and adult rabbit skeletal muscles. The results obtained show that the expression of the RyR3 isoform is developmentally regulated. In fact, RyR3 expression was detected in all muscles analysed at 2 and 15 days after birth while, in adult animals, it was restricted to a subset of muscles that includes diaphragm, masseter, pterygoideus, digastricus, and tongue. Interestingly, all of these muscles share a common embryonic origin being derived from the somitomeres or from the cephalic region of the embryo. Immunofluorescence analysis of rabbit skeletal muscle cross-sections showed that RyR3 staining was detected in all fibers of neonatal muscles. In contrast, in those adult muscles expressing RyR3 only a fraction of fibers was labelled. Staining of these muscles with antibodies against fast and slow myosins revealed a close correlation between expression of RyR3 and fibers expressing slow myosin isoform.

  13. The sodium channel Nav1.5a is the predominant isoform expressed in adult mouse dorsal root ganglia and exhibits distinct inactivation properties from the full-length Nav1.5 channel.

    PubMed

    Kerr, Niall C H; Gao, Zhan; Holmes, Fiona E; Hobson, Sally-Ann; Hancox, Jules C; Wynick, David; James, Andrew F

    2007-06-01

    Nav1.5 is the principal voltage-gated sodium channel expressed in heart, and is also expressed at lower abundance in embryonic dorsal root ganglia (DRG) with little or no expression reported postnatally. We report here the expression of Nav1.5 mRNA isoforms in adult mouse and rat DRG. The major isoform of mouse DRG is Nav1.5a, which encodes a protein with an IDII/III cytoplasmic loop reduced by 53 amino acids. Western blot analysis of adult mouse DRG membrane proteins confirmed the expression of Nav1.5 protein. The Na+ current produced by the Nav1.5a isoform has a voltage-dependent inactivation significantly shifted to more negative potentials (by approximately 5 mV) compared to the full-length Nav1.5 when expressed in the DRG neuroblastoma cell line ND7/23. These results imply that the alternatively spliced exon 18 of Nav1.5 plays a role in channel inactivation and that Nav1.5a is likely to make a significant contribution to adult DRG neuronal function.

  14. The expression of the truncated isoform of somatostatin receptor subtype 5 associates with aggressiveness in medullary thyroid carcinoma cells.

    PubMed

    Molè, Daniela; Gentilin, Erica; Ibañez-Costa, Alejandro; Gagliano, Teresa; Gahete, Manuel D; Tagliati, Federico; Rossi, Roberta; Pelizzo, Maria Rosa; Pansini, Giancarlo; Luque, Raúl M; Castaño, Justo P; degli Uberti, Ettore; Zatelli, Maria Chiara

    2015-11-01

    The truncated somatostatin receptor variant sst5TMD4 associates with increased invasiveness and aggressiveness in breast cancer. We previously found that sst5 activation may counteract sst2 selective agonist effects in a medullary thyroid carcinoma (MTC) cell line, the TT cells, and that sst5TMD4 is overexpressed in poorly differentiated thyroid cancers. The purpose of this study is to evaluate sst5TMD4 expression in a series of human MTC and to explore the functional role of sst5TMD4 in TT cells. We evaluated sst5TMD4 and sst5 expression in 36 MTC samples. Moreover, we investigated the role of sst5TMD4 in TT cells evaluating cell number, DNA synthesis, free cytosolic calcium concentration ([Ca(2+)]i), calcitonin and vascular endothelial growth factor levels, cell morphology, protein expression, and invasion. We found that in MTC the balance between sst5TMD4 and sst5 expression influences disease stage. sst5TMD4 overexpression in TT cells confers a greater growth capacity, blocks sst2 agonist-induced antiproliferative effects, modifies the cell phenotype, decreases E-cadherin and phosphorylated β-catenin levels, increases vimentin, total β-catenin and phosphorylated GSK3B levels (in keeping with the development of epithelial to mesenchymal transition), and confers a greater invasion capacity. This is the first evidence indicating that sst5TMD4 is expressed in human MTC cells, where it associates with more aggressive behavior, suggesting that sst5TMD4 might play a functionally relevant role.

  15. Regulation of Leishmania (L.) amazonensis protein expression by host T cell dependent responses: differential expression of oligopeptidase B, tryparedoxin peroxidase and HSP70 isoforms in amastigotes isolated from BALB/c and BALB/c nude mice.

    PubMed

    Teixeira, Priscila Camillo; Velasquez, Leonardo Garcia; Lepique, Ana Paula; de Rezende, Eloiza; Bonatto, José Matheus Camargo; Barcinski, Marcello Andre; Cunha-Neto, Edecio; Stolf, Beatriz Simonsen

    2015-02-01

    Leishmaniasis is an important disease that affects 12 million people in 88 countries, with 2 million new cases every year. Leishmania amazonensis is an important agent in Brazil, leading to clinical forms varying from localized (LCL) to diffuse cutaneous leishmaniasis (DCL). One interesting issue rarely analyzed is how host immune response affects Leishmania phenotype and virulence. Aiming to study the effect of host immune system on Leishmania proteins we compared proteomes of amastigotes isolated from BALB/c and BALB/c nude mice. The athymic nude mice may resemble patients with diffuse cutaneous leishmaniasis, considered T-cell hyposensitive or anergic to Leishmania's antigens. This work is the first to compare modifications in amastigotes' proteomes driven by host immune response. Among the 44 differentially expressed spots, there were proteins related to oxidative/nitrosative stress and proteases. Some correspond to known Leishmania virulence factors such as OPB and tryparedoxin peroxidase. Specific isoforms of these two proteins were increased in parasites from nude mice, suggesting that T cells probably restrain their posttranslational modifications in BALB/c mice. On the other hand, an isoform of HSP70 was increased in amastigotes from BALB/c mice. We believe our study may allow identification of potential virulence factors and ways of regulating their expression.

  16. Tunable protein synthesis by transcript isoforms in human cells.

    PubMed

    Floor, Stephen N; Doudna, Jennifer A

    2016-01-06

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

  17. Onapristone (ZK299) and mifepristone (RU486) regulate the messenger RNA and protein expression levels of the progesterone receptor isoforms A and B in the bovine endometrium.

    PubMed

    Rekawiecki, Robert; Kowalik, Magdalena K; Kotwica, Jan

    2015-08-01

    The aim of this study was to examine whether progesterone (P(4)) and its antagonists, onapristone (ZK299) and mifepristone (RU486), affect the levels of PGRA and PGRB messenger RNA (mRNA) and protein in the cow uterus which may be important in understanding whether the final physiological effect evoked by an antagonist depends on PGR isoform bound to the antagonist. Endometrial slices on Days 6 to 10 and 17 to 20 of the estrous cycle were treated for 6 or 24 hours for mRNA and protein expression analysis, respectively, with P4, ZK299, or RU486 at a dose of 10(-4), 10(-5), or 10(-6) M. In the samples on Days 6 to 10 of the estrous cycle, PGRAB mRNA was stimulated by P(4) (10(-4) M; P < 0.01) and RU486 (10(-6); P < 0.001) and was decreased by ZK299 (10(-5); P < 0.05). In contrast, PGRB mRNA was decreased by the all P(4) (P < 0.01) and ZK299 (P < 0.001) doses and by two of the RU486 doses (10(-4) M; P < 0.01 and 10(-5) M; P < 0.01). In samples on Days 17 to 20 of the estrous cycle, PGRAB mRNA was stimulated by RU486 (10(-5) M; P < 0.001). PGRB mRNA was decreased by P(4) (10(-4) and 10(-5) M; P < 0.001), ZK299 (10(-4) and 10(-5) M; P < 0.001), and RU486 (10(-4) M; P < 0.01 and 10(-6) M; P < 0.001) and was increased by ZK299 (10(-6) M; P < 0.001) and RU486 (10(-5) M; P < 0.001). In samples on Days 6 to 10 of the estrous cycle, PGRB protein levels were decreased (P < 0.05) by all three ZK299 doses and by two of the RU486 doses (10(-4) M; P < 0.05 and 10(-5) M; P < 0.01). In contrast, in samples on Days 17 to 20, both PGRA and PGRB protein levels were decreased by ZK299 stimulation (10(-5) M; P < 0.05 and 10(-5) M; P < 0.01, respectively), whereas only PGRA protein levels were increased by RU486 (10(-5) M; P < 0.01). Both ZK299 and RU486 may exhibit both agonist and antagonist properties depending on which receptor isoform they affect. As a result, an increase or decrease in the expression of a particular PGR isoform will be observed.

  18. Distinguishing between PrPC and PrPSc using small molecule reagents

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background/Introduction. The structural difference between PrPSc and PrPC is entirely conformational: they are isoforms. Both isoforms possess identical covalent structures and identical post-translational modifications. This means that the same amino acid can react differently with the same chemic...

  19. Distinguishing between PrPC and PrPSc using small molecule reagents(Abstract)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background/Introduction. The structural difference between PrPSc and PrPC is entirely conformational: they are isoforms. Both isoforms possess identical covalent structures and identical post-translational modifications. This means that the same amino acid can react differently with the same chemica...

  20. Small molecules and antibodies: a means of distinguishing between PrPC and PrPSc

    Technology Transfer Automated Retrieval System (TEKTRAN)

    PrPSc and PrPC are isoforms, since they possess identical covalent structures and identical post-translational modifications. The same amino acid may react differently with the same chemical reagent in an isoform-dependent manner. The site of covalent modification can be identified by mass spectrom...

  1. The expression profile of the major mouse SPO11 isoforms indicates that SPO11beta introduces double strand breaks and suggests that SPO11alpha has an additional role in prophase in both spermatocytes and oocytes.

    PubMed

    Bellani, Marina A; Boateng, Kingsley A; McLeod, Dianne; Camerini-Otero, R Daniel

    2010-09-01

    Both in mice and humans, two major SPO11 isoforms are generated by alternative splicing: SPO11alpha (exon 2 skipped) and SPO11beta. Thus, the alternative splicing event must have emerged before the mouse and human lineages diverged and was maintained during 90 million years of evolution, arguing for an essential role for both isoforms. Here we demonstrate that developmental regulation of alternative splicing at the Spo11 locus governs the sequential expression of SPO11 isoforms in male meiotic prophase. Protein quantification in juvenile mice and in prophase mutants indicates that early spermatocytes synthesize primarily SPO11beta. Estimation of the number of SPO11 dimers (betabeta/alphabeta/alphaalpha) in mutants in which spermatocytes undergo a normal number of double strand breaks but arrest in midprophase due to inefficient repair argues for a role for SPO11beta-containing dimers in introducing the breaks in leptonema. Expression kinetics in males suggested a role for SPO11alpha in pachytene/diplotene spermatocytes. Nevertheless, we found that both alternative transcripts can be detected in oocytes throughout prophase I, arguing against a male-specific function for this isoform. Altogether, our data support a role for SPO11alpha in mid- to late prophase, presumably acting as a topoisomerase, that would be conserved in male and female meiocytes.

  2. The polyphenol oxidase gene family in poplar: phylogeny, differential expression and identification of a novel, vacuolar isoform.

    PubMed

    Tran, Lan T; Constabel, C Peter

    2011-10-01

    Polyphenol oxidases (PPOs) are oxidative enzymes that convert monophenols and o-diphenols to o-quinones using molecular oxygen. The quinone products are highly reactive following tissue damage and can interact with cellular constituents and cause oxidative browning and cross-linking. The induction of PPO in some plants as a result of wounding, herbivore attack, or pathogen infection has implicated them in defense. However, PPO-like enzymes that act as specific hydroxylases, for example in lignan and pigment biosynthesis, have also been discovered. Here, we present the first genome-enabled analysis of a PPO gene family. The Populus trichocarpa genome was found to contain a minimum of nine complete PPO genes, and seven of these were characterized further. The PPO gene family includes both recently duplicated and divergent sequences that are 36-98% identical at the amino acid level. Gene expression profiling in poplar tissues and organs revealed that the PPO genes are all differentially expressed during normal development, but that only a small subset of PPO genes are significantly upregulated by wounding, methyl jasmonate or pathogen infection. Our studies also identified PtrPPO13, a novel PPO gene that is predicted to encode an N-terminal signal peptide. Transient expression of green fluorescent protein fusions demonstrated its localization to the vacuolar lumen. Together, our findings show that the poplar PPO family is diverse and is likely linked to diverse physiological functions.

  3. Basal and copper-induced expression of metallothionein isoform 1,2 and 3 genes in epithelial cancer cells: The role of tumor suppressor p53.

    PubMed

    Ostrakhovitch, E A; Song, Y P; Cherian, M G

    2016-05-01

    Metallothioneins (MTs) are a ubiquitous low-molecular weight, cysteine rich proteins with a high affinity for metal ions. The expression and induction of MTs have been associated with protection against DNA damage, oxidative stress, and apoptosis. Our past research had shown that p53 is an important factor in metal regulation of MTs. The present study was undertaken to explore further the interrelationship between p53 and MTs. We investigated whether silencing of p53 could affect expression pattern of basal and copper induced metallothioneins. The silencing of wild-type p53 (wt-p53) in epithelial breast cancer MCF7 cells affected the basal level of MT-2A RNA, whereas the levels of MT-1A and MT-1X RNA remained largely unchanged. The expression of MT-3 was undetectable in MCF7 with either functional or silenced p53. MCF7 cells with silenced wt-p53 failed to upregulate MT-2A in response to copper and showed a reduced sensitivity toward copper induced cell apoptotic death. Similarly in MCF7-E6 and MDA-MB-231 cells, the presence of inactive/mutated p53 halted MT-1A and MT-2A gene expression in response to copper. Constitutive expression of MT-3 RNA was detectable in the presence of mutated p53 (mtp53). Transient transfection of MDA-MB-231 cells with wt-p53 enabled copper induced upregulation of both MT-1A and MT-2A but not basal level of MT-2A, MT-1E, MT-1X and MT-3. Inactivation of p53 in HepG2 cells amplified the basal expression of studied MT isoforms, including MT-3, as well as copper-induced mRNA expression of MTs except MT-1H and MT-3. Presented data demonstrate a direct relation between p53 and MT-1A and MT-2A and they also indicate that wt-p53 might be a negative regulator of MT-3 in epithelial cancer cells.

  4. Contractile properties of motor units and expression of myosin heavy chain isoforms in rat fast-type muscle after volitional weight-lifting training.

    PubMed

    Łochyński, Dawid; Kaczmarek, Dominik; Mrówczyński, Włodzimierz; Warchoł, Wojciech; Majerczak, Joanna; Karasiński, Janusz; Korostyński, Michał; Zoladz, Jerzy A; Celichowski, Jan

    2016-10-01

    Dynamic resistance training increases the force and speed of muscle contraction, but little is known about modifications to the contractile properties of the main physiological types of motor units (MUs) that contribute to these muscle adaptations. Although the contractile profile of MU muscle fibers is tightly coupled to myosin heavy chain (MyHC) protein expression, it is not well understood if MyHC transition is a prerequisite for modifications to the contractile characteristics of MUs. In this study, we examined MU contractile properties, the mRNA expression of MyHC, parvalbumin, and sarcoendoplasmic reticulum Ca(2+) pump isoforms, as well as the MyHC protein content after 5 wk of volitional progressive weight-lifting training in the medial gastrocnemius muscle in rats. The training had no effect on MyHC profiling or Ca(2+)-handling protein gene expression. Maximum force increased in slow (by 49%) and fast (by 21%) MUs. Within fast MUs, the maximum force increased in most fatigue-resistant and intermediate but not most fatigable MUs. Twitch contraction time was shortened in slow and fast fatigue-resistant MUs. Twitch half-relaxation was shortened in fast most fatigue-resistant and intermediate MUs. The force-frequency curve shifted rightward in fast fatigue-resistant MUs. Fast fatigable MUs fatigued less within the initial 15 s while fast fatigue-resistant units increased the ability to potentiate the force within the first minute of the standard fatigue test. In conclusion, at the early stage of resistance training, modifications to the contractile characteristics of MUs appear in the absence of MyHC transition and the upregulation of Ca(2+)-handling genes.

  5. Functional characterization of EZH2β reveals the increased complexity of EZH2 isoforms involved in the regulation of mammalian gene expression

    PubMed Central

    2013-01-01

    Background Histone methyltransferase enhancer of zeste homologue 2 (EZH2) forms an obligate repressive complex with suppressor of zeste 12 and embryonic ectoderm development, which is thought, along with EZH1, to be primarily responsible for mediating Polycomb-dependent gene silencing. Polycomb-mediated repression influences gene expression across the entire gamut of biological processes, including development, differentiation and cellular proliferation. Deregulation of EZH2 expression is implicated in numerous complex human diseases. To date, most EZH2-mediated function has been primarily ascribed to a single protein product of the EZH2 locus. Results We report that the EZH2 locus undergoes alternative splicing to yield at least two structurally and functionally distinct EZH2 methyltransferases. The longest protein encoded by this locus is the conventional enzyme, which we refer to as EZH2α, whereas EZH2β, characterized here, represents a novel isoform. We find that EZH2β localizes to the cell nucleus, complexes with embryonic ectoderm development and suppressor of zeste 12, trimethylates histone 3 at lysine 27, and mediates silencing of target promoters. At the cell biological level, we find that increased EZH2β induces cell proliferation, demonstrating that this protein is functional in the regulation of processes previously attributed to EZH2α. Biochemically, through the use of genome-wide expression profiling, we demonstrate that EZH2β governs a pattern of gene repression that is often ontologically redundant from that of EZH2α, but also divergent for a wide variety of specific target genes. Conclusions Combined, these results demonstrate that an expanded repertoire of EZH2 writers can modulate histone code instruction during histone 3 lysine 27-mediated gene silencing. These data support the notion that the regulation of EZH2-mediated gene silencing is more complex than previously anticipated and should guide the design and interpretation of future

  6. Functional specificity of PMCA isoforms?

    PubMed

    Domi, Teuta; Di Leva, Francesca; Fedrizzi, Laura; Rimessi, Alessandro; Brini, Marisa

    2007-03-01

    In mammals, four different genes encode four PMCA isoforms. PMCA1 and PMCA4 are expressed ubiquitously. PMCA2 and PMCA3 are expressed prevalently in the central nervous systems. More than 30 variants are generated by mechanisms of alternative splicing. The physiological meaning of the existence of such elevated number of isoforms is not clear, but it would be plausible to relate it to the cell-specific demands of Ca2+ homeostasis. To characterize functional specificity of PMCA variants we have investigated two aspects: the effects of the overexpression of the different PMCA variants on cellular Ca2+ handling and the existence of possible isoform-specific interactions with partner proteins using a yeast two-hybrid technique. The four basic PMCA isoforms were coexpressed in CHO cells together with the Ca2+-sensitive recombinant photoprotein aequorin. The effects of their overexpression on Ca2+ homeostasis were monitored in the living cells. They had revealed that the ubiquitous isoforms 1 and 4 are less effective in reducing the Ca2+ peaks generated by cell stimulation as compared to the neuron-specific isoforms 2 and 3. To establish whether these differences were related to different and new physiological regulators of the pump, the 90 N-terminal residues of PMCA2 and PMCA4 have been used as baits for the search of molecular partners. Screening of a human brain cDNA library with the PMCA4 bait specified the epsilon-isoform of protein 14-3-3, whereas no 14-3-3 epsilon clone was obtained with the PMCA2 bait. Overexpression of PMCA4/14-3-3 epsilon (but not of PMCA2/14-3-3 epsilon) in HeLa cells together with targeted aequorins showed that the ability of the cells to export Ca2+ was impaired. Thus, the interaction with 14-3-3 epsilon inhibited PMCA4 but not PMCA2. The role of PMCA2 has been further characterized by Ca2+ measurements in cells overexpressing different splicing variants. The results indicated that the combination of alternative splicing at two different

  7. Glucocorticoid adrenal steroids and glucocorticoid-inducible kinase isoforms in the regulation of GluR6 expression

    PubMed Central

    Strutz-Seebohm, Nathalie; Seebohm, Guiscard; Shumilina, Ekaterina; Mack, Andreas F; Wagner, Hans-Joachim; Lampert, Angelika; Grahammer, Florian; Henke, Guido; Just, Lothar; Skutella, Thomas; Hollmann, Michael; Lang, Florian

    2005-01-01

    Generation of memory is enhanced during stress, an effect attributed to stimulation of neuronal learning by adrenal glucocorticoids. The glucocorticoid-dependent genes include the serum- and glucocorticoid-inducible kinase SGK1. SGK1 is activated through the phosphatidylinositol 3 kinase (PI3-kinase) pathway by growth factors such as insulin-like growth factor-1 (IGF1) or tumour growth factor β (TGF-β). Previously, a fourfold higher expression of SGK1 has been observed in fast-learning rats as compared with slow-learning rats. The mechanisms linking glucocorticoids or SGK1 with neuronal function have, however, remained elusive. We show here that treatment of mice with the glucocorticoid dexamethasone (238 μg day−1 for 8–20 days) enhances hippocampal expression of GluR6. Immunohistochemistry reveals significantly enhanced GluR6 protein abundance at neurones but not at astrocytes in mice. Immunohistochemistry and patch clamp on hippocampal neurones in primary culture reveal upregulation of GluR6 protein abundance and kainate-induced currents following treatment with dexamethasone (1 μm) and TGF-β (1 μm). In Xenopus oocytes expressing rat GluR6, coexpression of SGK1 strongly increases glutamate-induced current at least partially by increasing the abundance of GluR6 protein in the plasma membrane. The related kinases SGK2 and SGK3 similarly stimulate GluR6, but are less effective than SGK1. The observations point to a novel mechanism regulating GluR6 which contributes to the regulation of neuronal function by glucocorticoids. PMID:15774535

  8. The mRNA expression profile of metabolic genes relative to MHC isoform pattern in human skeletal muscles.

    PubMed

    Plomgaard, Peter; Penkowa, Milena; Leick, Lotte; Pedersen, Bente K; Saltin, Bengt; Pilegaard, Henriette

    2006-09-01

    The metabolic profile of rodent muscle is generally reflected in the myosin heavy chain (MHC) fiber-type composition. The present study was conducted to test the hypothesis that metabolic gene expression is not tightly coupled with MHC fiber-type composition for all genes in human skeletal muscle. Triceps brachii, vastus lateralis quadriceps, and soleus muscle biopsies were obtained from normally physically active, healthy, young male volunteers, because these muscles are characterized by different fiber-type compositions. As expected, citrate synthase and 3-hydroxyacyl dehydrogenase activity was more than twofold higher in soleus and vastus than in triceps. Contrary, phosphofructokinase and total lactate dehydrogenase (LDH) activity was approximately three- and twofold higher in triceps than in both soleus and vastus. Expression of metabolic genes was assessed by determining the mRNA content of a broad range of metabolic genes. The triceps muscle had two- to fivefold higher MHC IIa, phosphofructokinase, and LDH A mRNA content and two- to fourfold lower MHC I, lipoprotein lipase, CD36, hormone-sensitive lipase, and LDH B and hexokinase II mRNA than vastus lateralis or soleus. Interestingly, such mRNA differences were not evident for any of the genes encoding mitochondrial oxidative proteins, 3-hydroxyacyl dehydrogenase, carnitine palmitoyl transferase I, citrate synthase, alpha-ketogluterate dehydrogenase, and cytochrome c, nor for the transcriptional regulators peroxisome proliferator activator receptor gamma coactivator-1alpha, forkhead box O1, or peroxisome proliferator activator receptor-alpha. Thus the mRNA expression of genes encoding mitochondrial proteins and transcriptional regulators does not seem to be fiber type specific as the genes encoding glycolytic and lipid metabolism genes, which suggests that basal mRNA regulation of genes encoding mitochondrial proteins does not match the wide differences in mitochondrial content of these muscles.

  9. Expression dynamics of HSP90 and nitric oxide synthase (NOS) isoforms during heat stress acclimation in Tharparkar cattle

    NASA Astrophysics Data System (ADS)

    Bharati, Jaya; Dangi, S. S.; Bag, S.; Maurya, V. P.; Singh, G.; Kumar, P.; Sarkar, M.

    2017-03-01

    Six male Tharparkar cattle of 2-3 years old were selected for the study. After 15-day acclimation at thermoneutral zone (TNZ) in psychrometric chamber, animals were exposed at 42 °C for 6 h up to 23 days followed by 12 days of recovery period. Blood samples were collected during control period at TNZ (days 1, 5, and 12), after heat stress exposure (day 1, immediate heat stress acclimation (IHSA); days 2 to 10, short-term heat stress acclimation (STHSA); days 15 to 23, long-term heat stress acclimation (LTHSA); days 7 and 12, recovery period), and peripheral blood mononuclear cells (PBMCs) were isolated for RNA and protein extraction. The messenger RNA (mRNA) and protein expression in PBMCs were determined by qPCR and western blot, respectively. Samples at TNZ were taken as control. The mRNA expression of HSP90, iNOS, and eNOS was significantly upregulated (P < 0.05) on day 1 (ISHA) as compared to control, remained consistent during STHSA, again increased during LTHSA, and finally reduced to basal level during recovery period. The protein expression of HSP90, iNOS, and eNOS were akin to their transcript pattern. PBMC culture study was conducted to study transcriptional abundance of HSP90, iNOS, and eNOS at different temperature-time combinations. The present findings indicate that HSP90, iNOS, and eNOS could possibly play an important role in mitigating thermal insults and confer thermotolerance during long-term heat stress exposure in Tharparkar cattle.

  10. Effects of quercetin on intracavernous pressure and expression of nitrogen synthase isoforms in arterial erectile dysfunction rat model

    PubMed Central

    Zhang, Yueyang; Huang, Changting; Liu, Shaoming; Bai, Jianqi; Fan, Xiaojing; Guo, Jun; Jia, Yingyu; Zhang, Zhijie; Chen, Xiaojun; Jia, Yusen; Zhang, Ping; Wang, Bin; Zhang, Xiuju

    2015-01-01

    Object: Oxidative stress involved in the regulation of arterial erectile dysfunction (A-ED). Previously report have indicated that quercetin have an antioxidant effect. In the current study, we have established the rats’ model for study the therapeutic effect of quercetin on A-ED and further investigated the molecular mechanism of action. Methods: Wistar rats were divided into sham group, A-ED group, A-ED group with low dose of quercetin, and A-ED group with high dose of quercetin. Intracavernous pressure (ICP) and mean arterial pressure (MBp) are two important indicators used for evaluation the A-ED. The changes of ICP and MBp were determined by cavernous nerve electrostimulation after treatment of quercetin at indicated doses. The expression of nitric oxide synthase (NOS) subtypes was detected by RT-PCR and Western blotting. Results: Our results indicated that ICP was significantly reduced in A-ED rats model compared with sham group, and was significantly increased after quercetin treatment (P < 0.01), while no significant effect on the MBp. The data also showed that sGC inhibitor ODQ and NOS inhibitor LNNA can significantly inhibited the ICP which induced by quercetin. These results suggest that NO-cGMP signaling pathway plays a crucial role in A-ED. Then, we found that the mRNA and protein levels of eNOS were significantly reduced in A-ED group compared with sham group. After treated with quercetin may cause the eNOS RNA and protein were significantly up-regulated (P < 0.01), showing a dose-dependent effect. iNOS expression have a certain degree of increased after quercetin treatment. nNOS expression was not significantly increased before and after treated with quercetin. In a word, quercetin can improved the A-ED by up-regulated ICP, which related to up-regulation of NO-cGMP signaling pathway. Conclusion: Preliminary results of this study suggested that quercetin protected expression and function of eNOS in cavernous endothelial cells, and restored part of

  11. Phase 1/2 open-label dose-escalation study of plasmid DNA expressing two isoforms of hepatocyte growth factor in patients with painful diabetic peripheral neuropathy.

    PubMed

    Ajroud-Driss, Senda; Christiansen, Mark; Allen, Jeffrey A; Kessler, John A

    2013-06-01

    This study aimed to evaluate the safety and preliminary efficacy of intramuscular injections of plasmid DNA (VM202) expressing two isoforms of hepatocyte growth factor (HGF) in subjects with painful diabetic peripheral neuropathy (PDPN). Twelve patients in three cohorts (4, 8, and 16 mg) received two sets of VM202 injections separated by two weeks. Safety and tolerability were evaluated and the visual analog scale (VAS), the short form McGill questionnaire (SF-MPQ), and the brief pain inventory for patients with diabetic peripheral neuropathy (BPI-DPN) measured pain level throughout 12 months after treatment. No serious adverse events (AEs) were observed. The mean VAS was reduced from baseline by 47.2% (P = 0.002) at 6 months and by 44.1% (P = 0.005) at 12 months after treatment. The VAS scores for the 4, 8, and 16 mg dose cohorts at 6 months follow-up decreased in a dose-responsive manner, by 21% (P = 0.971), 53% (P = 0.014), and 62% (P = 0.001), respectively. The results with the BPI-DPN and SF-MPQ showed patterns similar to the VAS scores. In conclusion, VM202 treatment appeared to be safe, well tolerated, and sufficient to provide long term symptomatic relief and improvement in the quality of life in patients with PDPN.

  12. Properties and Expression of Na+/K+-ATPase α-Subunit Isoforms in the Brain of the Swamp Eel, Monopterus albus, Which Has Unusually High Brain Ammonia Tolerance

    PubMed Central

    Chen, Xiu L.; Wee, Nicklaus L. J. E.; Hiong, Kum C.; Ong, Jasmine L. Y.; Chng, You R.; Ching, Biyun; Wong, Wai P.; Chew, Shit F.; Ip, Yuen K.

    2013-01-01

    The swamp eel, Monopterus albus, can survive in high concentrations of ammonia (>75 mmol l−1) and accumulate ammonia to high concentrations in its brain (∼4.5 µmol g−1). Na+/K+-ATPase (Nka) is an essential transporter in brain cells, and since NH4+ can substitute for K+ to activate Nka, we hypothesized that the brain of M. albus expressed multiple forms of Nka α-subunits, some of which might have high K+ specificity. Thus, this study aimed to clone and sequence the nka α-subunits from the brain of M. albus, and to determine the effects of ammonia exposure on their mRNA expression and overall protein abundance. The effectiveness of NH4+ to activate brain Nka from M. albus and Mus musculus was also examined by comparing their Na+/K+-ATPase and Na+/NH4+-ATPase activities over a range of K+/NH4+ concentrations. The full length cDNA coding sequences of three nkaα (nkaα1, nkaα3a and nkaα3b) were identified in the brain of M. albus, but nkaα2 expression was undetectable. Exposure to 50 mmol l−1 NH4Cl for 1 day or 6 days resulted in significant decreases in the mRNA expression of nkaα1, nkaα3a and nkaα3b. The overall Nka protein abundance also decreased significantly after 6 days of ammonia exposure. For M. albus, brain Na+/NH4+-ATPase activities were significantly lower than the Na+/K+-ATPase activities assayed at various NH4+/K+ concentrations. Furthermore, the effectiveness of NH4+ to activate Nka from the brain of M. albus was significantly lower than that from the brain of M. musculus, which is ammonia-sensitive. Hence, the (1) lack of nkaα2 expression, (2) high K+ specificity of K+ binding sites of Nkaα1, Nkaα3a and Nkaα3b, and (3) down-regulation of mRNA expression of all three nkaα isoforms and the overall Nka protein abundance in response to ammonia exposure might be some of the contributing factors to the high brain ammonia tolerance in M. albus. PMID:24391932

  13. Cloning, characterization and gene expression of a metallothionein isoform in the edible cockle Cerastoderma edule after cadmium or mercury exposure.

    PubMed

    Paul-Pont, Ika; Gonzalez, Patrice; Montero, Natalia; de Montaudouin, Xavier; Baudrimont, Magalie

    2012-01-01

    Metallothionein (MT) genes encode crucial metal-binding proteins ubiquitously expressed in living organisms and which play important roles in homeostasis of essential metals and detoxification processes. Here, the molecular organization of the first metallothionein gene of the edible cockle Cerastoderma edule and its expression after cadmium (Cd) or mercury (Hg) exposures were determined. The resulting sequence (Cemt1) exhibits unusual features. The full length cDNA encodes a protein of 73 amino acids with nine classical Cys-X((1-3))-Cys motifs, but also one Cys-Cys not generally found in molluscan MT. Moreover, characterization of the molecular organization of the Cemt1 gene revealed two different alleles (A1 and A2) with length differences due to large deletion events in their intronic sequences involving direct Short Interspersed repeated Elements (SINE), while their exonic sequences were identical. To our knowledge, such large excision mechanisms have never before been reported in a bivalve gene sequence. After 10 days of Cd exposure at environmentally relevant doses, quantitative real-time PCR revealed a strong induction of Cemt1 in gills of C. edule. Surprisingly, neither induction of the Cemt1 gene nor of MT protein was shown after Hg exposure, despite the fact that this organism is able to bioaccumulate a high amount of this trace metal which is theoretically one of the most powerful inducers of MT biosynthesis.

  14. The effect of PrP(Sc) accumulation on inflammatory gene expression within sheep peripheral lymphoid tissue.

    PubMed

    Gossner, Anton G; Hopkins, John

    2015-12-31

    Accumulation of the misfolded prion protein, PrP(Sc) in the central nervous system (CNS) is strongly linked to progressive neurodegenerative disease. For many transmissible spongiform encephalopathies (TSEs), peripheral lymphoid tissue is an important site of PrP(Sc) amplification but without gross immunological consequence. Susceptible VRQ homozygous New Zealand Cheviot sheep were infected with SSBP/1 scrapie by inoculation in the drainage area of the prescapular lymph nodes. The earliest time that PrP(Sc) was consistently detected by immunohistology in these nodes was D50 post infection. This transcriptomic study of lymph node taken before (D10) and after (D50) the detection of PrP(Sc), aimed to identify the genes and physiological pathways affected by disease progression within the nodes as assessed by PrP(Sc) detection. Affymetrix Ovine Gene arrays identified 75 and 80 genes as differentially-expressed at D10 and D50, respectively, in comparison with control sheep inoculated with uninfected brain homogenate. Approximately 70% of these were repressed at each time point. RT-qPCR analysis of seven genes showed statistically significant correlation with the array data, although the results for IL1RN and TGIF were different between the two technologies. The ingenuity pathway analysis (IPA) and general low level of repression of gene expression in lymphoid tissue, including many inflammatory genes, contrasts with the pro-inflammatory and pro-apoptotic events that occur within the CNS at equivalent stages of disease progression as assessed by PrP(Sc) accumulation.

  15. Post-translational modification of the NKG2D ligand RAET1G leads to cell surface expression of a glycosylphosphatidylinositol-linked isoform.

    PubMed

    Ohashi, Maki; Eagle, Robert A; Trowsdale, John

    2010-05-28

    NKG2D is an important activating receptor on lymphocytes. In human, it interacts with two groups of ligands: the major histocompatibility complex class I chain-related A/B (MICA/B) family and the UL-16 binding protein (ULBP) family, also known as retinoic acid early transcript (RAET1). MIC proteins are membrane-anchored, but all of the ULBP/RAET1 proteins, except for RAET1E and RAET1G, are glycosylphosphatidylinositol (GPI)-anchored. To address the reason for these differences we studied the association of RAET1G with the membrane. Using epitope-tagged RAET1G protein in conjunction with antibodies to different parts of the molecule and in pulse-chase experiments, we showed that the C terminus of the protein was cleaved soon after protein synthesis. Endoglycosidase H and peptide N-glycosidase treatment and cell surface immunoprecipitation indicated that most of the protein stayed in the endoplasmic reticulum, but some of the cleaved form was modified in the Golgi and transported to the cell surface. We examined the possibility of GPI anchoring of the protein in three ways: (i) Phosphatidylinositol (PI)-specific phospholipase C released the PI-linked form of the protein. (ii) The surface expression pattern of RAET1G decreased in cells defective in GPI anchoring through mutant GPI-amidase. (iii) Site-directed mutagenesis, to disrupt residues predicted to facilitate GPI-anchoring, resulted in diminished surface expression of RAET1G. Thus, a form of RAET1G is GPI-anchored, in line with most other ULBP/RAET1 family proteins. The cytoplasmic tail and transmembrane domains appear to result from gene duplication and frameshift mutation. Together with our previous results, our data suggest that RAET1G is regulated post-translationally to produce a GPI-anchored isoform.

  16. Post-translational Modification of the NKG2D Ligand RAET1G Leads to Cell Surface Expression of a Glycosylphosphatidylinositol-linked Isoform*

    PubMed Central

    Ohashi, Maki; Eagle, Robert A.; Trowsdale, John

    2010-01-01

    NKG2D is an important activating receptor on lymphocytes. In human, it interacts with two groups of ligands: the major histocompatibility complex class I chain-related A/B (MICA/B) family and the UL-16 binding protein (ULBP) family, also known as retinoic acid early transcript (RAET1). MIC proteins are membrane-anchored, but all of the ULBP/RAET1 proteins, except for RAET1E and RAET1G, are glycosylphosphatidylinositol (GPI)-anchored. To address the reason for these differences we studied the association of RAET1G with the membrane. Using epitope-tagged RAET1G protein in conjunction with antibodies to different parts of the molecule and in pulse-chase experiments, we showed that the C terminus of the protein was cleaved soon after protein synthesis. Endoglycosidase H and peptide N-glycosidase treatment and cell surface immunoprecipitation indicated that most of the protein stayed in the endoplasmic reticulum, but some of the cleaved form was modified in the Golgi and transported to the cell surface. We examined the possibility of GPI anchoring of the protein in three ways: (i) Phosphatidylinositol (PI)-specific phospholipase C released the PI-linked form of the protein. (ii) The surface expression pattern of RAET1G decreased in cells defective in GPI anchoring through mutant GPI-amidase. (iii) Site-directed mutagenesis, to disrupt residues predicted to facilitate GPI-anchoring, resulted in diminished surface expression of RAET1G. Thus, a form of RAET1G is GPI-anchored, in line with most other ULBP/RAET1 family proteins. The cytoplasmic tail and transmembrane domains appear to result from gene duplication and frameshift mutation. Together with our previous results, our data suggest that RAET1G is regulated post-translationally to produce a GPI-anchored isoform. PMID:20304922

  17. Short-term strength training and the expression of myostatin and IGF-I isoforms in rat muscle and tendon: differential effects of specific contraction types.

    PubMed

    Heinemeier, K M; Olesen, J L; Schjerling, P; Haddad, F; Langberg, H; Baldwin, K M; Kjaer, M

    2007-02-01

    In skeletal muscle, an increased expression of insulin like growth factor-I isoforms IGF-IEa and mechano-growth factor (MGF) combined with downregulation of myostatin is thought to be essential for training-induced hypertrophy. However, the specific effects of different contraction types on regulation of these factors in muscle are still unclear, and in tendon the functions of myostatin, IGF-IEa, and MGF in relation to training are unknown. Female Sprague-Dawley rats were subjected to 4 days of concentric, eccentric, or isometric training (n = 7-9 per group) of the medial gastrocnemius, by stimulation of the sciatic nerve during general anesthesia. mRNA levels for myostatin, IGF-IEa, and MGF in muscle and Achilles' tendon were measured by real-time RT-PCR. Muscle myostatin mRNA decreased in response to all types of training (2- to 8-fold) (P < 0.05), but the effect of eccentric training was greater than concentric and isometric training (P < 0.05). In tendon, myostatin mRNA was detected, but no changes were seen after exercise. IGF-IEa and MGF increased in muscle (up to 15-fold) and tendon (up to 4-fold) in response to training (P < 0.01). In tendon no difference was seen between training types, but in muscle the effect of eccentric training was greater than concentric training for both IGF-IEa and MGF (P < 0.05), and for IGF-IEa isometric training had greater effect than concentric (P < 0.05). The results indicate a possible role for IGF-IEa and MGF in adaptation of tendon to training, and the combined changes in myostatin and IGF-IEa/MGF expression could explain the important effect of eccentric actions for muscle hypertrophy.

  18. Neuroplastin Isoform Np55 Is Expressed in the Stereocilia of Outer Hair Cells and Required for Normal Outer Hair Cell Function

    PubMed Central

    Zeng, Wei-Zheng; Grillet, Nicolas; Dewey, James B.; Trouillet, Alix; Krey, Jocelyn F.; Barr-Gillespie, Peter G.; Oghalai, John S.

    2016-01-01

    Neuroplastin (Nptn) is a member of the Ig superfamily and is expressed in two isoforms, Np55 and Np65. Np65 regulates synaptic transmission but the function of Np55 is unknown. In an N-ethyl-N-nitrosaurea mutagenesis screen, we have now generated a mouse line with an Nptn mutation that causes deafness. We show that Np55 is expressed in stereocilia of outer hair cells (OHCs) but not inner hair cells and affects interactions of stereocilia with the tectorial membrane. In vivo vibrometry demonstrates that cochlear amplification is absent in Nptn mutant mice, which is consistent with the failure of OHC stereocilia to maintain stable interactions with the tectorial membrane. Hair bundles show morphological defects as the mutant mice age and while mechanotransduction currents can be evoked in early postnatal hair cells, cochlea microphonics recordings indicate that mechanontransduction is affected as the mutant mice age. We thus conclude that differential splicing leads to functional diversification of Nptn, where Np55 is essential for OHC function, while Np65 is implicated in the regulation of synaptic function. SIGNIFICANCE STATEMENT Amplification of input sound signals, which is needed for the auditory sense organ to detect sounds over a wide intensity range, depends on mechanical coupling of outer hair cells to the tectorial membrane. The current study shows that neuroplastin, a member of the Ig superfamily, which has previously been linked to the regulation of synaptic plasticity, is critical to maintain a stable mechanical link of outer hair cells with the tectorial membrane. In vivo recordings demonstrate that neuroplastin is essential for sound amplification and that mutation in neuroplastin leads to auditory impairment in mice. PMID:27581460

  19. Inhibition of insulin receptor gene expression and insulin signaling by fatty acid: interplay of PKC isoforms therein.

    PubMed

    Dey, Debleena; Mukherjee, Mohua; Basu, Dipanjan; Datta, Malabika; Roy, Sib Sankar; Bandyopadhyay, Arun; Bhattacharya, Samir

    2005-01-01

    Fatty acids are known to play a key role in promoting the loss of insulin sensitivity causing insulin resistance and type 2 diabetes. However, underlying mechanism involved here is still unclear. Incubation of rat skeletal muscle cells with palmitate followed by I(125)- insulin binding to the plasma membrane receptor preparation demonstrated a two-fold decrease in receptor occupation. In searching the cause for this reduction, we found that palmitate inhibition of insulin receptor (IR) gene expression effecting reduced amount of IR protein in skeletal muscle cells. This was followed by the inhibition of insulin-stimulated IRbeta tyrosine phosphorylation that consequently resulted inhibition of insulin receptor substrate 1 (IRS 1) and IRS 1 associated phosphatidylinositol-3 kinase (PI3 Kinase), phosphoinositide dependent kinase-1 (PDK 1) phosphorylation. PDK 1 dependent phosphorylation of PKCzeta and Akt/PKB were also inhibited by palmitate. Surprisingly, although PKCepsilon phosphorylation is PDK1 dependent, palmitate effected its constitutive phosphorylation independent of PDK1. Time kinetics study showed translocation of palmitate induced phosphorylated PKCepsilon from cell membrane to nuclear region and its possible association with the inhibition of IR gene transcription. Our study suggests one of the pathways through which fatty acid can induce insulin resistance in skeletal muscle cell.

  20. Comparison of the QuantiGene 2.0 Assay and Real-Time RT-PCR in the Detection of p53 Isoform mRNA Expression in Formalin-Fixed Paraffin-Embedded Tissues- A Preliminary Study

    PubMed Central

    Morten, Brianna C.; Scott, Rodney J.; Avery-Kiejda, Kelly A.

    2016-01-01

    p53 is expressed as multiple smaller isoforms whose functions in cancer are not well understood. The p53 isoforms demonstrate abnormal expression in different cancers, suggesting they are important in modulating the function of full-length p53 (FLp53). The quantification of relative mRNA expression has routinely been performed using real-time PCR (qPCR). However, there are serious limitations when detecting p53 isoforms using this method, particularly for formalin-fixed paraffin-embedded (FFPE) tissues. The use of FFPE tumours would be advantageous to correlate expression of p53 isoforms with important clinical features of cancer. One alternative method of RNA detection is the hybridization-based QuantiGene 2.0 Assay, which has been shown to be advantageous for the detection of RNA from FFPE tissues. In this pilot study, we compared the QuantiGene 2.0 Assay to qPCR for the detection of FLp53 and its isoform Δ40p53 in matched fresh frozen (FF) and FFPE breast tumours. FLp53 mRNA expression was detected using qPCR in FF and FFPE tissues, but Δ40p53 mRNA was only detectable in FF tissues. Similar results were obtained for the QuantiGene 2.0 Assay. FLp53 relative mRNA expression was shown to be strongly correlated between the two methods (R2 = 0.9927, p = 0.0031) in FF tissues, however Δ40p53 was not (R2 = 0.4429, p = 0.3345). When comparing the different methods for the detection of FLp53 mRNA from FFPE and FF samples, no correlation (R2 = 0.0002, p = 0.9863) was shown using the QuantiGene 2.0 Assay, and in contrast, the level of expression was highly correlated between the two tissues using qPCR (R2 = 0.8753, p = 0.0644). These results suggest that both the QuantiGene 2.0 Assay and qPCR methods are inadequate for the quantification of Δ40p53 mRNA in FFPE tissues. Therefore, alternative methods of RNA detection and quantification are required to study the relative expression of Δ40p53 in FFPE samples. PMID:27832134

  1. Transgenic expression of medicago truncatula PR10 and PR5 promoters in alfalfa shows pathogen-induced up-regulation of transgene expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetic modification of alfalfa to introduce novel traits requires promoters for controlling gene expression. Promoters that are constitutively activated for expression of genes that enhance disease resistance pose a great energy load on the plant and exert a strong selective pressure on the pathoge...

  2. Structural evolution and tissue-specific expression of tetrapod-specific second isoform of secretory pathway Ca{sup 2+}-ATPase

    SciTech Connect

    Pestov, Nikolay B.; Dmitriev, Ruslan I.; Kostina, Maria B.; Korneenko, Tatyana V.; Shakhparonov, Mikhail I.; Modyanov, Nikolai N.

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer Full-length secretory pathway Ca-ATPase (SPCA2) cloned from rat duodenum. Black-Right-Pointing-Pointer ATP2C2 gene (encoding SPCA2) exists only in genomes of Tetrapoda. Black-Right-Pointing-Pointer Rat and pig SPCA2 are expressed in intestines, lung and some secretory glands. Black-Right-Pointing-Pointer Subcellular localization of SPCA2 may depend on tissue type. Black-Right-Pointing-Pointer In rat duodenum, SPCA2 is localized in plasma membrane-associated compartments. -- Abstract: Secretory pathway Ca-ATPases are less characterized mammalian calcium pumps than plasma membrane Ca-ATPases and sarco-endoplasmic reticulum Ca-ATPases. Here we report analysis of molecular evolution, alternative splicing, tissue-specific expression and subcellular localization of the second isoform of the secretory pathway Ca-ATPase (SPCA2), the product of the ATP2C2 gene. The primary structure of SPCA2 from rat duodenum deduced from full-length transcript contains 944 amino acid residues, and exhibits 65% sequence identity with known SPCA1. The rat SPCA2 sequence is also highly homologous to putative human protein KIAA0703, however, the latter seems to have an aberrant N-terminus originating from intron 2. The tissue-specificity of SPCA2 expression is different from ubiquitous SPCA1. Rat SPCA2 transcripts were detected predominantly in gastrointestinal tract, lung, trachea, lactating mammary gland, skin and preputial gland. In the newborn pig, the expression profile is very similar with one remarkable exception: porcine bulbourethral gland gave the strongest signal. Upon overexpression in cultured cells, SPCA2 shows an intracellular distribution with remarkable enrichment in Golgi. However, in vivo SPCA2 may be localized in compartments that differ among various tissues: it is intracellular in epidermis, but enriched in plasma membranes of the intestinal epithelium. Analysis of SPCA2 sequences from various vertebrate species argue that ATP2C2

  3. The Fungal Pathogen Moniliophthora perniciosa Has Genes Similar to Plant PR-1 That Are Highly Expressed during Its Interaction with Cacao

    PubMed Central

    Vidal, Ramon O.; do Prado, Paula F.V.; Reis, Osvaldo; Baroni, Renata M.; Franco, Sulamita F.; Mieczkowski, Piotr; Pereira, Gonçalo A.G.; Mondego, Jorge M.C.

    2012-01-01

    The widespread SCP/TAPS superfamily (SCP/Tpx-1/Ag5/PR-1/Sc7) has multiple biological functions, including roles in the immune response of plants and animals, development of male reproductive tract in mammals, venom activity in insects and reptiles and host invasion by parasitic worms. Plant Pathogenesis Related 1 (PR-1) proteins belong to this superfamily and have been characterized as markers of induced defense against pathogens. This work presents the characterization of eleven genes homologous to plant PR-1 genes, designated as MpPR-1, which were identified in the genome of Moniliophthora perniciosa, a basidiomycete fungus responsible for causing the devastating witches' broom disease in cacao. We describe gene structure, protein alignment and modeling analyses of the MpPR-1 family. Additionally, the expression profiles of MpPR-1 genes were assessed by qPCR in different stages throughout the fungal life cycle. A specific expression pattern was verified for each member of the MpPR-1 family in the conditions analyzed. Interestingly, some of them were highly and specifically expressed during the interaction of the fungus with cacao, suggesting a role for the MpPR-1 proteins in the infective process of this pathogen. Hypothetical functions assigned to members of the MpPR-1 family include neutralization of plant defenses, antimicrobial activity to avoid competitors and fruiting body physiology. This study provides strong evidence on the importance of PR-1-like genes for fungal virulence on plants. PMID:23029323

  4. The fungal pathogen Moniliophthora perniciosa has genes similar to plant PR-1 that are highly expressed during its interaction with cacao.

    PubMed

    Teixeira, Paulo J P L; Thomazella, Daniela P T; Vidal, Ramon O; do Prado, Paula F V; Reis, Osvaldo; Baroni, Renata M; Franco, Sulamita F; Mieczkowski, Piotr; Pereira, Gonçalo A G; Mondego, Jorge M C

    2012-01-01

    The widespread SCP/TAPS superfamily (SCP/Tpx-1/Ag5/PR-1/Sc7) has multiple biological functions, including roles in the immune response of plants and animals, development of male reproductive tract in mammals, venom activity in insects and reptiles and host invasion by parasitic worms. Plant Pathogenesis Related 1 (PR-1) proteins belong to this superfamily and have been characterized as markers of induced defense against pathogens. This work presents the characterization of eleven genes homologous to plant PR-1 genes, designated as MpPR-1, which were identified in the genome of Moniliophthora perniciosa, a basidiomycete fungus responsible for causing the devastating witches' broom disease in cacao. We describe gene structure, protein alignment and modeling analyses of the MpPR-1 family. Additionally, the expression profiles of MpPR-1 genes were assessed by qPCR in different stages throughout the fungal life cycle. A specific expression pattern was verified for each member of the MpPR-1 family in the conditions analyzed. Interestingly, some of them were highly and specifically expressed during the interaction of the fungus with cacao, suggesting a role for the MpPR-1 proteins in the infective process of this pathogen. Hypothetical functions assigned to members of the MpPR-1 family include neutralization of plant defenses, antimicrobial activity to avoid competitors and fruiting body physiology. This study provides strong evidence on the importance of PR-1-like genes for fungal virulence on plants.

  5. Expression of recombinant West Nile virus prM protein fused to an affinity tag for use as a diagnostic antigen.

    PubMed

    Setoh, Y X; Hobson-Peters, J; Prow, N A; Young, P R; Hall, R A

    2011-07-01

    Previous studies have concluded that the Flavivirus prM protein is a suitable viral antigen to distinguish serologically between infections with closely related Flaviviruses (Cardosa et al., 2002). To express the recombinant West Nile virus (WNV) prM antigen fused to a suitable affinity tag for purification, a series of prM-His-tag and prM-V5-tag fusion proteins were generated. Analysis of the prM-His-tag fusion proteins revealed that either prM epitopes were disrupted or the His-tag was not presented properly depending on the location of the His tag and the presence of the prM transmembrane domains in these constructs. This identified domains critical for proper folding of prM, and arrangements that allowed the correct presentation of the His-tag. However, the inclusion of the V5 epitope tag fused to the C terminus of prM allowed formation of the authentic antigenic structure of prM and the proper presentation of the V5 epitope. Capture of tagged recombinant WNV(NY99) prM antigen to the solid phase with anti-V5 antibody in ELISA enabled the detection of prM-specific antibodies in WNV(NY99)-immune horse serum, confirming its potential as a useful diagnostic reagent.

  6. An energy-rich diet enhances expression of Na(+)/H(+) exchanger isoform 1 and 3 messenger RNA in rumen epithelium of goat.

    PubMed

    Yang, W; Shen, Z; Martens, H

    2012-01-01

    Rumen epithelial Na(+)/H(+) exchanger (NHE) catalyzes the exchange of extracellular Na(+) for intracellular H(+). Thus, it is of importance in the maintenance of Na and pH homeostasis of rumen epithelial cells. We have tested the hypothesis that an increase in energy and protein intake induces alterations of NHE isoform 1, 2, and 3 (NHE1, NHE1, and NHE3, respectively) mRNA abundance in the rumen epithelium of goats. Goats (n = 26) were randomly allocated to 2 experiments (n = 16 in Exp. 1, and n = 10 in Exp. 2) and fed either peanut straw ad libitum [PNS, n = 8 in Exp. 1, and n = 5 in Exp. 2; 600 kJ of ME/(kg(0.75)·d)] or PNS + concentrate [CF, n = 8 in Exp. 1, and n = 5 in Exp. 2; 1,000 kJ of ME/(kg(0.75)·d)] for 42 d. Concentrate (400 g/d) was given daily (0800 to 1700 h) in 4 equal portions at 3-h intervals. In Exp. 1, the goats were euthanized 2 h after the last portion of concentrate was fed, and in Exp. 2, the goats were euthanized after a fasting period of 16 h. In Exp. 1, goats in the CF treatment exhibited a greater ruminal short-chain fatty acid (SCFA) concentration (140.6 ± 1.30 mM) compared with those in the PNS treatment (114.3 ± 3.11 mM; P < 0.001), and pH decreased from 6.9 ± 0.09 to 5.9 ± 0.04 (P < 0.001). Correspondingly, the mRNA expression of NHE1 and NHE3 in the rumen epithelium was greater by 20% (P = 0.041) and 25% (P = 0.043) for goats in the CF treatment than for those in the PNS treatment. However, in Exp. 2, 16 h of fasting abolished differences in ruminal SCFA concentration, pH, and NHE mRNA expression between goats in the CF and PNS treatments. In both Exp. 1 and 2, a positive correlation was observed between ruminal SCFA concentration and expression of mRNA in NHE1 and NHE3, whereas expression was negatively correlated with ruminal pH. In in vitro studies with isolated rumen epithelial cells from goats fed dried grass, exposure to pH of 6.8 or to 20 mM SCFA increased (P < 0.01) NHE1 and NHE3 mRNA expression, as compared with

  7. Expression of autocrine prolactin and the short isoform of prolactin receptor are associated with inflammatory response and apoptosis in monocytes stimulated with Mycobacterium bovis proteins.

    PubMed

    López-Rincón, Gonzalo; Mancilla, Raúl; Pereira-Suárez, Ana L; Martínez-Neri, Priscila A; Ochoa-Zarzosa, Alejandra; Muñoz-Valle, José Francisco; Estrada-Chávez, Ciro

    2015-06-01

    Increased levels of prolactin (PRL) have recently been associated with carcinogenesis and the exacerbation of autoimmune diseases, and might be involved in the progression of tuberculosis (TB). To investigate the relationship between PRL and prolactin receptor (PRLr) expression with inflammatory response and apoptosis in monocytes, we used THP-1 cells stimulated with antigens of the Mycobacterium bovis AN5 strain culture filtrate protein (CFP-M. bovis). Western blot (WB), real-time Polymerase chain reaction (PCR), and immunocytochemistry were performed to identify both PRL and PRLr molecules. PRL bioactivity and proinflammatory cytokine detection were assessed. The results showed that PRL and PRLr messenger RNA (mRNA) were synthesized in THP-1 monocytes induced with CFP-M. bovis at peaks of 176- and 404-fold, respectively. PRL forms of 60 and 80kDa and PRLr isoforms of 40, 50, and 65kDa were also identified as time-dependent, while 60-kDa PRL, as well as 40-, and 50-kDa PRLr, were found as soluble forms in culture media and later in the nucleus of THP-1 monocytes. PRL of 60kDa released by monocytes exhibited bioactivity in Nb2 cells, and both synthesized PRL and synthesized PRLr were related with nitrite and proinflammatory cytokine levels proapoptotic activity in CFP-M. bovis-induced monocytes. Our results suggest the overexpression of a full-autocrine loop of PRL and PRLr in monocytes that enhances the inflammatory response and apoptosis after priming with M. bovis antigens.

  8. Age-dependent decline in density of human nerve and spinal ganglia neurons expressing the α3 isoform of Na/K-ATPase

    PubMed Central

    Romanovsky, Dmitry; Mrak, Robert E.; Dobretsov, Maxim

    2015-01-01

    Ambulatory instability and falls are a major source of morbidity in the elderly. Age-related loss of tendon reflexes is a major contributing factor to this morbidity, and deterioration of the afferent limb of the stretch reflex is a potential contributing factor to such age-dependent loss of tendon reflexes. To evaluate this, we assessed the number and distribution of muscle spindle afferent fibers in human sacral spinal ganglia (S1) and tibial nerve samples obtained at autopsy, using immunohistochemical staining for the α3 isoform of Na+,K+-ATPase (α3NKA), a marker of muscle spindle afferents. Across all age groups, an average of 26±4% of myelinated fibers of tibial nerve and 17±2% of ganglion neuronal profiles were α3NKA-positive (n=8 per group). Subject age explained 85% of the variability in these counts. The relative frequency of α3NKA-labeled fibers/neurons starts to decline during the 5th decade of life, approaching half that of young adult values in 65-year-old subjects. At all ages, α3NKA-positive neurons were among the largest of spinal ganglia neurons. However, as compared to younger subjects, the population of α3NKA-positive neurons from advanced-age subjects showed diminished numbers of large (both moderately and strongly labeled), and medium-sized (strongly labeled) profiles. Considering the critical significance of ion transport by NKA for neuronal activity, our data suggest that functional impairment and, also, most likely atrophy and/or degeneration of muscle spindle afferents, are mechanisms underlying loss of tendon reflexes with age. The larger and more strongly α3NKA-expressing spindle afferents appear to be proportionally more vulnerable. PMID:26386295

  9. c-Jun NH2-terminal kinase-related Na+/H+ exchanger isoform 1 activation controls hexokinase II expression in benzo(a)pyrene-induced apoptosis.

    PubMed

    Huc, Laurence; Tekpli, Xavier; Holme, Jørn A; Rissel, Mary; Solhaug, Anita; Gardyn, Claire; Le Moigne, Gwénaelle; Gorria, Morgane; Dimanche-Boitrel, Marie-Thérèse; Lagadic-Gossmann, Dominique

    2007-02-15

    Regulation of the balance between survival, proliferation, and apoptosis on carcinogenic polycyclic aromatic hydrocarbon (PAH) exposure is still poorly understood and more particularly the role of physiologic variables, including intracellular pH (pH(i)). Although the involvement of the ubiquitous pH(i) regulator Na(+)/H(+) exchanger isoform 1 (NHE1) in tumorigenesis is well documented, less is known about its role and regulation during apoptosis. Our previous works have shown the primordial role of NHE1 in carcinogenic PAH-induced apoptosis. This alkalinizing transporter was activated by an early CYP1-dependent H(2)O(2) production, subsequently promoting mitochondrial dysfunction leading to apoptosis. The aim of this study was to further elucidate how NHE1 was activated by benzo(a)pyrene (BaP) and what the downstream events were in the context of apoptosis. Our results indicate that the mitogen-activated protein kinase kinase 4/c-Jun NH(2)-terminal kinase (MKK4/JNK) pathway was a link between BaP-induced H(2)O(2) production and NHE1 activation. This activation, in combination with BaP-induced phosphorylated p53, promoted mitochondrial superoxide anion production, supporting the existence of a common target for NHE1 and p53. Furthermore, we showed that the mitochondrial expression of glycolytic enzyme hexokinase II (HKII) was decreased following a combined action of NHE1 and p53 pathways, thereby enhancing the BaP-induced apoptosis. Taken together, our findings suggest that, on BaP exposure, MKK4/JNK targets NHE1 with consequences on HKII protein, which might thus be a key protein during carcinogenic PAH apoptosis.

  10. Altered microtubule-associated tau messenger RNA isoform expression in livers of griseofulvin- and 3,5-diethoxycarbonyl-1, 4-dihydrocollidine-treated mice.

    PubMed

    Kenner, L; Zatloukal, K; Stumptner, C; Eferl, R; Denk, H

    1999-03-01

    Tau proteins belong to the family of microtubule-associated proteins (MAPs), which so far have been mostly detected in neuronal cells. Different domains on the protein serve different functions. By alternative splicing, several mRNAs and tau isoforms are created from one gene, which contain these functionally important domains to various degrees, and thus differ in their microtubule-related properties. In the present article, several novel observations are reported. Tau mRNA and proteins have been identified and further characterized in mouse liver. It is shown on the basis of mRNA determinations that at least three tau isoforms differing particularly with respect to their amino-terminal domains are present in mouse liver. The major and predominant isoform (isoform 1) lacks portions encoded by exons 2 and 3, which are responsible for cross-talk of microtubules with their environment ("projection domain"). Moreover, mRNA encoding tau protein with four repeats of the microtubule binding domain predominate in embryonal as well as adult mouse liver in contrast to brain, in which a shift from the predominant three-repeat isoform to the four-repeat isoform characterizes the transition from the embryonic to the adult stage. Intoxication with griseofulvin (GF) or 3,5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) significantly affects in a reversible manner the levels of tau mRNA as well as isoform ratios in mouse liver, but not in mouse brain. Tau mRNAs are significantly increased in intoxicated mouse livers. Moreover, a shift to isoform 1 lacking exons 2 and 3 occurs. However, the increase in liver tau protein was less than expected from increased mRNA levels, which could be the result of translational or posttranslational regulation. The consequences on microtubular function are as yet unclear, but impairment can be expected because the overexpressed tau mRNA isoform lacks the domain that mediates interaction of microtubules with their environment. On the other hand, the

  11. Two Isoforms of the RNA Binding Protein, Coding Region Determinant-binding Protein (CRD-BP/IGF2BP1), Are Expressed in Breast Epithelium and Support Clonogenic Growth of Breast Tumor Cells.

    PubMed

    Fakhraldeen, Saja A; Clark, Rod J; Roopra, Avtar; Chin, Emily N; Huang, Wei; Castorino, John; Wisinski, Kari B; Kim, TaeWon; Spiegelman, Vladimir S; Alexander, Caroline M

    2015-05-22

    CRD-BP/IGF2BP1 has been characterized as an "oncofetal" RNA binding protein typically highly expressed in embryonic tissues, suppressed in normal adult tissues, but induced in many tumor types. In this study, we show that adult breast tissues express ubiquitous but low levels of CRD-BP protein and mRNA. Although CRD-BP mRNA expression is induced in breast tumor cells, levels remain ∼1000-fold lower than in embryonic tissues. Despite low expression levels, CRD-BP is required for clonogenic growth of breast cancer cells. We reveal that because the most common protein isoform in normal adult breast and breast tumors has an N-terminal deletion (lacking two RNA recognition motif (RRM) domains) and is therefore missing antibody epitopes, CRD-BP expression has been under-reported by previous studies. We show that a CRD-BP mutant mouse strain retains expression of the shorter transcript (ΔN-CRD-BP), which originates in intron 2, suggesting that the impact of complete ablation of this gene in mice is not yet known. Either the full-length CRD-BP or the N-terminally truncated version can rescue the clonogenicity of CRD-BP knockdown breast cancer cells, suggesting that clonogenic function is served by either CRD-BP isoform. In summary, although CRD-BP expression levels are low in breast cancer cells, this protein is necessary for clonogenic activity.

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

    PubMed

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

    2015-05-01

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

  13. Molecular cloning and expression of human UDP-d-Xylose:proteoglycan core protein beta-d-xylosyltransferase and its first isoform XT-II.

    PubMed

    Götting, C; Kuhn, J; Zahn, R; Brinkmann, T; Kleesiek, K

    2000-12-08

    Human UDP-d-xylose:proteoglycan core protein beta-d-xylosyltransferase (EC 2.4.2.26, XT-I) initiates the biosynthesis of glycosaminoglycan chains in proteoglycans by transferring xylose from UDP-xylose to specific serine residues of the core protein. Based on the partial amino acid sequence of the purified enzyme from human JAR choriocarcinoma cell culture supernatant we isolated a cDNA encoding XT-I using the degenerate reverse transcriptase-polymerase chain reaction method. This enzyme, which is involved in chondroitin sulfate, heparan sulfate, heparin and dermatan sulfate biosynthesis, belongs to a novel family of glycosyltransferases with no homology to proteins known so far. 5' and 3'-RACE were performed to isolate a novel cDNA fragment of 3726 bp with a single open reading frame encoding at least 827 amino acid residues with a molecular mass of 91 kDa. The human XT-I gene was located on chromosome 16p13.1 using radiation hybrid mapping, and extracts from CHO-K1 cells transfected with the XT-I cDNA in an expression vector exhibited marked XT activity. A new 3608 bp cDNA fragment encoding a protein of 865 amino acid residues was also isolated by PCR using degenerate primers based on the amino acid sequence of human XT-I. The amino acid sequence of this XT-II isoform displayed 55% identity to the human XT-I. The XT-II gene was located on chromosome 17q21.3-17q22, and the exon/intron structure of the 15 kb gene was determined. RT-PCR analyses of XT-I and XT-II mRNA from various tissues confirmed that both XT-I and XT-II transcripts are ubiquitously expressed in the human tissues, although with different levels of transcription. Furthermore, the cDNAs encoding XT-I and XT-II from rat were cloned. The deduced amino acid sequences of rat xylosyltransferases displayed 94% identity to the corresponding human enzyme.

  14. INK4 locus of the tumor-resistant rodent, the naked mole rat, expresses a functional p15/p16 hybrid isoform.

    PubMed

    Tian, Xiao; Azpurua, Jorge; Ke, Zhonghe; Augereau, Adeline; Zhang, Zhengdong D; Vijg, Jan; Gladyshev, Vadim N; Gorbunova, Vera; Seluanov, Andrei

    2015-01-27

    The naked mole rat (Heterocephalus glaber) is a long-lived and tumor-resistant rodent. Tumor resistance in the naked mole rat is mediated by the extracellular matrix component hyaluronan of very high molecular weight (HMW-HA). HMW-HA triggers hypersensitivity of naked mole rat cells to contact inhibition, which is associated with induction of the INK4 (inhibitors of cyclin dependent kinase 4) locus leading to cell-cycle arrest. The INK4a/b locus is among the most frequently mutated in human cancer. This locus encodes three distinct tumor suppressors: p15(INK4b), p16(INK4a), and ARF (alternate reading frame). Although p15(INK4b) has its own ORF, p16(INK4a) and ARF share common second and third exons with alternative reading frames. Here, we show that, in the naked mole rat, the INK4a/b locus encodes an additional product that consists of p15(INK4b) exon 1 joined to p16(INK4a) exons 2 and 3. We have named this isoform pALT(INK4a/b) (for alternative splicing). We show that pALT(INK4a/b) is present in both cultured cells and naked mole rat tissues but is absent in human and mouse cells. Additionally, we demonstrate that pALT(INK4a/b) expression is induced during early contact inhibition and upon a variety of stresses such as UV, gamma irradiation-induced senescence, loss of substrate attachment, and expression of oncogenes. When overexpressed in naked mole rat or human cells, pALT(INK4a/b) has stronger ability to induce cell-cycle arrest than either p15(INK4b) or p16(INK4a). We hypothesize that the presence of the fourth product, pALT(INK4a/b) of the INK4a/b locus in the naked mole rat, contributes to the increased resistance to tumorigenesis of this species.

  15. Characterization and heterologous expression of a PR-1 protein from traps of the carnivorous plant Nepenthes mirabilis.

    PubMed

    Buch, Franziska; Pauchet, Yannick; Rott, Matthias; Mithöfer, Axel

    2014-04-01

    Carnivorous plants capture and digest prey to obtain additional nutrients. Therefore, different trapping mechanisms were developed in different species. Plants of the genus Nepenthes possess pitfall-traps filled with a digestive fluid, which is secreted by the plants themselves. This pitcher fluid is composed of various enzymes to digest the captured prey. Besides hydrolytic enzymes, defense-related proteins have been identified in the fluid. The present study describes the identification and heterologous expression of a pathogenesis-related protein, NmPR-1, from pitchers of Nepenthes mirabilis with features that are unusual for PR-1 proteins. In particular, it was proven to be highly glycosylated and, furthermore, it exhibited antibacterial instead of antifungal activities. These properties are probably due to the specific environment of the pitcher fluid.

  16. A Point Mutation in DNA Polymerase β (POLB) Gene Is Associated with Increased Progesterone Receptor (PR) Expression and Intraperitoneal Metastasis in Gastric Cancer

    PubMed Central

    Tan, Xiaohui; Wu, Xiaoling; Ren, Shuyang; Wang, Hongyi; Li, Zhongwu; Alshenawy, Weaam; Li, Wenmei; Cui, Jiantao; Luo, Guangbin; Siegel, Robert S.; Fu, Sidney W.; Lu, Youyong

    2016-01-01

    Increased expression of progesterone receptor (PR) has been reported in gastric cancer (GC). We have previously identified a functional T889C point mutation in DNA polymerase beta (POLB), a DNA repair gene in GC. To provide a detailed analysis of molecular changes associated with the mutation, human cDNA microarrays focusing on 18 signal transduction pathways were used to analyze differential gene expression profiles between GC tissues with T889C mutant in POLB gene and those with wild type. Among the differentially expressed genes, notably, PR was one of the significantly up-regulated genes in T889C mutant POLB tissues, which were subsequently confirmed in POLB gene transfected AGS cell line. Interestingly, patients with T889C mutation and PR positivity were associated with higher incidence of intraperitoneal metastasis (IM). In vitro studies indicate that PR expression was upregulated in AGS cell line when transfected with T889C mutant expression vector. Cotransfection of T889C mutant allele and PR gene induced cell migration in the cell line. These data demonstrated that T889C mutation-associated PR overexpression results in increased IM. Therefore, T889C mutation-associated PR overexpression may serve as a biomarker for an adverse prognosis for human GC. PMID:27471563

  17. Characterization of endogenous human promyelocytic leukemia isoforms.

    PubMed

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

    2006-06-15

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

  18. Tumorigenic properties of alternative osteopontin isoforms in mesothelioma

    SciTech Connect

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

    2009-05-08

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

  19. Propagation of RML prions in mice expressing PrP devoid of GPI anchor leads to formation of a novel, stable prion strain.

    PubMed

    Mahal, Sukhvir Paul; Jablonski, Joseph; Suponitsky-Kroyter, Irena; Oelschlegel, Anja Maria; Herva, Maria Eugenia; Oldstone, Michael; Weissmann, Charles

    2012-01-01

    PrP(C), a host protein which in prion-infected animals is converted to PrP(Sc), is linked to the cell membrane by a GPI anchor. Mice expressing PrP(C) without GPI anchor (tgGPI⁻ mice), are susceptible to prion infection but accumulate anchorless PrP(Sc) extra-, rather than intracellularly. We investigated whether tgGPI⁻ mice could faithfully propagate prion strains despite the deviant structure and location of anchorless PrP(Sc). We found that RML and ME7, but not 22L prions propagated in tgGPI⁻ brain developed novel cell tropisms, as determined by the Cell Panel Assay (CPA). Surprisingly, the levels of proteinase K-resistant PrP(Sc) (PrP(res)) in RML- or ME7-infected tgGPI⁻ brain were 25-50 times higher than in wild-type brain. When returned to wild-type brain, ME7 prions recovered their original properties, however RML prions had given rise to a novel prion strain, designated SFL, which remained unchanged even after three passages in wild-type mice. Because both RML PrP(Sc) and SFL PrP(Sc) are stably propagated in wild-type mice we propose that the two conformations are separated by a high activation energy barrier which is abrogated in tgGPI⁻ mice.

  20. PrP-dependent cell adhesion in N2a neuroblastoma cells.

    PubMed

    Mangé, Alain; Milhavet, Ollivier; Umlauf, David; Harris, David; Lehmann, Sylvain

    2002-03-13

    The cellular isoform of prion protein (PrP(C)) is a ubiquitous glycoprotein expressed by most tissues and with a biological function yet to be determined. Here, we have used a neuroblastoma cell model to investigate the involvement of PrP in cell adhesion. Incubation of single cell suspension induced cell-cell adhesion and formation of cell aggregates. Interestingly, cells overexpressing PrP exhibit increased cation-independent aggregation. Aggregation was reduced after phosphatidylinositol-specific phospholipase C release of the protein and by pre-incubation of cells with an antibody raised against the N-terminal part of PrP(C). Our paradigm allows the study of the function of PrP as an intercellular adhesion molecule and a cell surface ligand or receptor.

  1. Bovine PrP expression levels in transgenic mice influence transmission characteristics of atypical bovine spongiform encephalopathy.

    PubMed

    Wilson, Rona; Hart, Patricia; Piccardo, Pedro; Hunter, Nora; Casalone, Cristina; Baron, Thierry; Barron, Rona M

    2012-05-01

    Until recently, transmissible spongiform encephalopathy (TSE) disease in cattle was thought to be caused by a single agent strain, bovine spongiform encephalopathy (BSE) (classical BSE or BSE-C). However, due to the initiation of a large-scale surveillance programme throughout Europe, two atypical BSE strains, bovine amyloidotic spongiform encephalopathy (BASE, also named BSE-L) and BSE-H have since been discovered. These atypical BSE isolates have been previously transmitted to a range of transgenic mouse models overexpressing PrP from different species at different levels, on a variety of genetic backgrounds. To control for genetic background and expression level in the analysis of these isolates, we performed here a comprehensive comparison of the neuropathological and molecular properties of all three BSE agents (BASE, BSE-C and BSE-H) upon transmission into the same gene-targeted transgenic mouse line expressing the bovine prion protein (Bov6) and a wild-type control of the same genetic background. Significantly, upon challenge with these BSE agents, we found that BASE did not produce shorter survival times in these mice compared with BSE-C, contrary to previous studies using overexpressing bovine transgenic mice. Amyloid plaques were only present in mice challenged with atypical BSE and neuropathological features, including intensity of PrP deposition in the brain and severity of vacuolar degeneration were less pronounced in BASE compared with BSE-C-challenged mice.

  2. Expression of a Serine Protease Gene prC Is Up-Regulated by Oxidative Stress in the Fungus Clonostachys rosea: Implications for Fungal Survival

    PubMed Central

    Liu, Wen-Jing; Zhou, Wei; Tao, Nan; Tu, Hui-Hui; Huang, Xiao-Wei; Yang, Jin-Kui; Zhang, Ke-Qin

    2010-01-01

    Background Soil fungi face a variety of environmental stresses such as UV light, high temperature, and heavy metals. Adaptation of gene expression through transcriptional regulation is a key mechanism in fungal response to environmental stress. In Saccharomyces cerevisiae, the transcription factors Msn2/4 induce stress-mediated gene expression by binding to the stress response element. Previous studies have demonstrated that the expression of extracellular proteases is up-regulated in response to heat shock in fungi. However, the physiological significance of regulation of these extracellular proteases by heat shock remains unclear. The nematophagous fungus Clonostachys rosea can secret an extracellular serine protease PrC during the infection of nematodes. Since the promoter of prC has three copies of the stress response element, we investigated the effect of environmental stress on the expression of prC. Methodology/Principal Findings Our results demonstrated that the expression of prC was up-regulated by oxidants (H2O2 or menadione) and heat shock, most likely through the stress response element. After oxidant treatment or heat shock, the germination of conidia in the wild type strain was significantly higher than that in the prC mutant strain in the presence of nematode cuticle. Interestingly, the addition of nematode cuticle significantly attenuated the production of reactive oxygen species (ROS) induced by oxidants and heat shock in the wild type strain, but not in prC mutant strain. Moreover, low molecule weight (<3 kD) degradation products of nematode cuticle suppressed the inhibitory effect of conidial germination induced by oxidants and heat shock. Conclusions/Significance These results indicate that PrC plays a protective role in oxidative stress in C. rosea. PrC degrades the nematode cuticle to produce degradation products, which in turn offer a protective effect against oxidative stress by scavenging ROS. Our study reveals a novel strategy for fungi to

  3. Dopamine-Induced Apoptosis of Lactotropes Is Mediated by the Short Isoform of D2 Receptor

    PubMed Central

    Radl, Daniela Betiana; Ferraris, Jimena; Boti, Valeria; Seilicovich, Adriana; Sarkar, Dipak Kumar; Pisera, Daniel

    2011-01-01

    Dopamine, through D2 receptor (D2R), is the major regulator of lactotrope function in the anterior pituitary gland. Both D2R isoforms, long (D2L) and short (D2S), are expressed in lactotropes. Although both isoforms can transduce dopamine signal, they differ in the mechanism that leads to cell response. The administration of D2R agonists, such as cabergoline, is the main pharmacological treatment for prolactinomas, but resistance to these drugs exists, which has been associated with alterations in D2R expression. We previously reported that dopamine and cabergoline induce apoptosis of lactotropes in primary culture in an estrogen-dependent manner. In this study we used an in vivo model to confirm the permissive action of estradiol in the apoptosis of anterior pituitary cells induced by D2R agonists. Administration of cabergoline to female rats induced apoptosis, measured by Annexin-V staining, in anterior pituitary gland from estradiol-treated rats but not from ovariectomized rats. To evaluate the participation of D2R isoforms in the apoptosis induced by dopamine we used lactotrope-derived PR1 cells stably transfected with expression vectors encoding D2L or D2S receptors. In the presence of estradiol, dopamine induced apoptosis, determined by ELISA and TUNEL assay, only in PR1-D2S cells. To study the role of p38 MAPK in apoptosis induced by D2R activation, anterior pituitary cells from primary culture or PR1-D2S were incubated with an inhibitor of the p38 MAPK pathway (SB203850). SB203580 blocked the apoptotic effect of D2R activation in lactotropes from primary cultures and PR1-D2S cells. Dopamine also induced p38 MAPK phosphorylation, determined by western blot, in PR1-D2S cells and estradiol enhanced this effect. These data suggest that, in the presence of estradiol, D2R agonists induce apoptosis of lactotropes by their interaction with D2S receptors and that p38 MAPK is involved in this process. PMID:21464994

  4. Transcription variants of the prostate-specific PrLZ gene and their interaction with 14-3-3 proteins

    SciTech Connect

    Wang, Ruoxiang; He, Hui; Sun, Xiaojuan; Xu, Jianchun; Marshall, Fray F.; Zhau, Haiyen; Chung, Leland W.K.; Fu, Haian; He, Dalin

    2009-11-20

    We have reported isolation and characterization of the prostate-specific and androgen-regulated PrLZ gene abnormally expressed in prostate cancer. PrLZ is a potential biomarker for prostate cancer and a candidate oncogene promoting cell proliferation and survival in prostate cancer cells. A full delineation of the PrLZ gene and its gene products may provide clues to the mechanisms regulating its expression and function. In this report, we identified three additional exons in the PrLZ gene and recognized five transcript variants from alternative splicing that could be detected by RT-PCR and Western blotting. Structural comparison demonstrated that the PrLZ proteins are highly conserved among species. PrLZ contains multiple potential sites for interaction with other proteins. We used mammalian two-hybrid assays to demonstrate that PrLZ isoforms interact with 14-3-3 proteins, and multiple sites in the PrLZ may be involved in the interaction. Alternative splicing may contribute to abnormally enhanced PrLZ levels in prostate cancer, and interaction with 14-3-3 proteins may be a mechanism by which PrLZ promotes cell proliferation and survival during prostate cancer development and progression. This information is a valuable addition to the investigation of the oncogenic properties of the PrLZ gene.

  5. Type I (RI) and type II (RII) receptors for transforming growth factor-beta isoforms are expressed subsequent to transforming growth factor-beta ligands during excisional wound repair.

    PubMed Central

    Gold, L. I.; Sung, J. J.; Siebert, J. W.; Longaker, M. T.

    1997-01-01

    Transforming growth factor (TGF)-beta isoforms (TGF-beta 1, -beta 2, and -beta 3) regulate cell growth and differentiation and have critical regulatory roles in the process of tissue repair and remodeling. Signal transduction for TGF-beta function is transmitted by a heteromeric complex of receptors consisting of two serine/threonine kinase transmembrane proteins (RI and RII). We have previously shown that each TGF-beta isoform is widely expressed in a distinct spatial and temporal pattern throughout the processes of excisional and incisional wound repair. As the presence of TGF-beta receptors determines cellular responsiveness, we have currently examined, by immunohistochemistry, the localization of RI (ALK-1, ALK-5) and RII throughout repair of full-thickness excisional wounds up to 21 days after wounding. The expression of RI (ALK-5) and RII co-localized in both the unwounded and wounded skin and was present in the same cell types as TGF-beta ligands. However, immunoreactivity for TGF-beta receptors, throughout repair, occurred 1 to 5 days later than TGF-beta isoform immunostaining. This implies that the presence of TGF-beta ligands may up-regulate TGF-beta receptors for function and/or may reflect a lag due to local processing of latent TGF-beta. As observed for the immunohistochemical localization of TGF-beta isoforms in unwounded skin, RI and RII were expressed throughout the four layers of the epidermis, showing a wavy pattern of slight to moderate immunostaining, and hair follicles, sweat glands, and sebaceous glands were moderately immunoreactive. The extracellular matrix, fibroblasts, and blood vessels in the dermis were not immunoreactive. After injury, as observed for TGF-beta ligands, RI and RII expression was increased in the epidermis adjacent to the wound and the epithelium migrating over the wound was completely devoid of TGF-beta receptor immunoreactivity until re-epithelialization was completed by day 7 after wounding. The dermis was only

  6. Absolute Quantification of Endogenous Ras Isoform Abundance

    PubMed Central

    Mageean, Craig J.; Griffiths, John R.; Smith, Duncan L.; Clague, Michael J.; Prior, Ian A.

    2015-01-01

    Ras proteins are important signalling hubs situated near the top of networks controlling cell proliferation, differentiation and survival. Three almost identical isoforms, HRAS, KRAS and NRAS, are ubiquitously expressed yet have differing biological and oncogenic properties. In order to help understand the relative biological contributions of each isoform we have optimised a quantitative proteomics method for accurately measuring Ras isoform protein copy number per cell. The use of isotopic protein standards together with selected reaction monitoring for diagnostic peptides is sensitive, robust and suitable for application to sub-milligram quantities of lysates. We find that in a panel of isogenic SW48 colorectal cancer cells, endogenous Ras proteins are highly abundant with ≥260,000 total Ras protein copies per cell and the rank order of isoform abundance is KRAS>NRAS≥HRAS. A subset of oncogenic KRAS mutants exhibit increased total cellular Ras abundance and altered the ratio of mutant versus wild type KRAS protein. These data and methodology are significant because Ras protein copy number is required to parameterise models of signalling networks and informs interpretation of isoform-specific Ras functional data. PMID:26560143

  7. Structural isoforms of the circadian neuropeptide PDF expressed in the optic lobes of the cricket Gryllus bimaculatus: immunocytochemical evidence from specific monoclonal antibodies.

    PubMed

    Honda, Takeshi; Matsushima, Ayami; Sumida, Kazunori; Chuman, Yoshiro; Sakaguchi, Kazuyasu; Onoue, Hitoshi; Meinertzhagen, Ian A; Shimohigashi, Yasuyuki; Shimohigashi, Miki

    2006-11-20

    Pigment-dispersing factor (PDF) is an 18-mer peptide that acts as a principal neurotransmitter of the insect circadian clock. Our previous study, utilizing anti-Uca beta-PDH polyclonal antibody (pAb) to immunolabel the optic lobe of the cricket Gryllus bimaculatus, suggested the existence of an alternative PDF-like peptide in the outer cells of the first neuropile, or lamina (La), which were much less immunoreactive than the inner cells of the second neuropile, the medulla (Me). To obtain structural information about such a PDF-like peptide, we prepared 10 anti-Gryllus PDF monoclonal (mAb) and pAb antibodies and analyzed their detailed epitope specificities. The PDFMe and PDFLa inner cells and their axonal projections were clearly immunoreactive to all these antibodies, revealing the widespread immunocytochemical organization of the PDF system in the optic lobe, as seen previously with anti-Uca beta-PDH pAb and anti-Gryllus PDF mAb, the epitope structures of which were also clarified in this study. The lamina outer cells, which we found lacked a target pdf mRNA, displayed specific immunoreactivities, indicating that the cells contain a distinct PDF-like peptide possessing both N- and C-terminal structures. These cells were not immunolabeled by some other monoclonal antibodies, however, implying that the PDFLa outer cells have a PDF isoform peptide devoid of Asn at positions 6 and 16. This isoform was also identified in a varicose arborization in the lamina. These results suggest not only the structure of the peptide, but also the possibility of additional functions of this novel PDF isoform.

  8. Trehalose metabolism in the blue crab Callinectes sapidus: isolation of multiple structural cDNA isoforms of trehalose-6-phosphate synthase and their expression in muscles.

    PubMed

    Shi, Q; Chung, J Sook

    2014-02-15

    Adult blue crab Callinectes sapidus exhibit behavioral and ecological dimorphisms: females migrating from the low salinity water to the high salinity area vs. males remaining in the same areas. The flesh basal muscle of the swimming paddle shows a dimorphic color pattern in that levator (Lev) and depressor (Dep) of females tend to be much darker than those of males, while both genders have the same light colored remoter (Rem) and promoter (Pro). The full-length cDNA sequence of four structural isoforms of trehalose-6-phosphate synthase (TPS) is isolated from chela muscles of an adult female, C. sapidus. Two isoforms of the C. sapidus TPS encode functional domains of TPS and trehalose-6-phosphorylase (TPP) in tandem as a fused gene product of Escherichia coli Ost A and Ost B. The other two isoforms contain only a single TPS domain. In both males and females, the darker (Lev+Dep) muscles exhibit greater amounts of trehalose, TPS and trehalase activities than the light colored (Rem+Pro). The fact that adult females show higher levels of trehalase activity in the basal muscles and of glucose in Lev+Dep than those of adult males suggests that there may be a metabolic dimorphism. Moreover, the involvement of trehalose in energy metabolism that was examined under the condition of strenuous swimming activity mimicked in adult females demonstrates the intrinsic trehalose metabolism in Lev+Dep, which subsequently results in hemolymphatic hyperglycemia and hyperlactemia. Our data support that trehalose serves as an additional carbohydrate source of hemolymphatic hyperglycemia in this species. Behavioral and ecological dimorphisms of C. sapidus adults may be supported by a functional dimorphism in energy metabolism.

  9. VEGFA splicing: divergent isoforms regulate spermatogonial stem cell maintenance

    PubMed Central

    Sargent, Kevin M.; Clopton, Debra T.; Lu, Ningxia; Pohlmeier, William E.

    2015-01-01

    Despite being well-known for regulating angiogenesis in both normal and tumorigenic environments, vascular endothelial growth factor A (VEGFA) has been recently implicated in male fertility, namely in the maintenance of spermatogonial stem cells (SSC). The VEGFA gene can be spliced into multiple distinct isoforms that are either angiogenic or antiangiogenic in nature. Although studies have demonstrated the alternative splicing of VEGFA, including the divergent roles of the two isoform family types, many investigations do not differentiate between them. Data concerning VEGFA in the mammalian testis are limited, but the various angiogenic isoforms appear to promote seminiferous cord formation and to form a gradient across which cells may migrate. Treatment with either antiangiogenic isoforms of VEGFA or with inhibitors to angiogenic signaling impair these processes. Serendipitously, expression of KDR, the primary receptor for both types of VEGFA isoforms, was observed on male germ cells. These findings led to further investigation of the way that VEGFA elicits avascular functions within testes. Following treatment of donor perinatal male mice with either antiangiogenic VEGFA165b or angiogenic VEGFA164 isoforms, seminiferous tubules were less colonized following transplantation with cells from VEGFA165b-treated donors. Thus, VEGFA165b and possibly other antiangiogenic isoforms of VEGFA reduce SSC number either by promoting premature differentiation, inducing cell death, or by preventing SSC formation. Thus, angiogenic isoforms of VEGFA are hypothesized to promote SSC self-renewal, and the divergent isoforms are thought to balance one another to maintain SSC homeostasis in vivo. PMID:26553653

  10. The function of Drosophila p53 isoforms in apoptosis

    PubMed Central

    Zhang, B; Rotelli, M; Dixon, M; Calvi, B R

    2015-01-01

    The p53 protein is a major mediator of the cellular response to genotoxic stress and is a crucial suppressor of tumor formation. In a variety of organisms, p53 and its paralogs, p63 and p73, each encode multiple protein isoforms through alternative splicing, promoters, and translation start sites. The function of these isoforms in development and disease are still being defined. Here, we evaluate the apoptotic potential of multiple isoforms of the single p53 gene in the genetic model Drosophila melanogaster. Most previous studies have focused on the p53A isoform, but it has been recently shown that a larger p53B isoform can induce apoptosis when overexpressed. It has remained unclear, however, whether one or both isoforms are required for the apoptotic response to genotoxic stress. We show that p53B is a much more potent inducer of apoptosis than p53A when overexpressed. Overexpression of two newly identified short isoforms perturbed development and inhibited the apoptotic response to ionizing radiation. Analysis of physiological protein expression indicated that p53A is the most abundant isoform, and that both p53A and p53B can form a complex and co-localize to sub-nuclear compartments. In contrast to the overexpression results, new isoform-specific loss-of-function mutants indicated that it is the shorter p53A isoform, not full-length p53B, that is the primary mediator of pro-apoptotic gene transcription and apoptosis after ionizing radiation. Together, our data show that it is the shorter p53A isoform that mediates the apoptotic response to DNA damage, and further suggest that p53B and shorter isoforms have specialized functions. PMID:25882045

  11. Role of Progesterone Receptor Isoforms in Regulation of Cell Adhesion and Apoptosis

    DTIC Science & Technology

    2002-06-01

    AD Award Number: DAMD17-01-1-0507 TITLE: Role of Progesterone Receptor Isoforms in Regulation of Cell Adhesion and Apoptosis PRINCIPAL...1 Jun 01 - 31 May 02) 4. TITLE AND SUBTITLE Role of Progesterone Receptor Isoforms in Regulation of Cell Adhesion and Apoptosis 6. AUTHOR(S...information) Progesterone receptors (PR) and estrogen receptors (ER) are important prognostic indicators in breast cancer. We believe that PR, in addition to

  12. Characterization of the isoforms of type IIb sodium-dependent phosphate cotransporter (Slc34a2) in yellow catfish, Pelteobagrus fulvidraco, and their vitamin D3-regulated expression under low-phosphate conditions.

    PubMed

    Chen, Pei; Huang, Yanqing; Bayir, Abdulkadir; Wang, Chunfang

    2017-02-01

    In this study, two isoforms slc34a2 genes (type IIb sodium-dependent phosphate cotransporter), slc34a2a2 and slc34a2b, were cloned from intestine and kidney of yellow catfish (Pelteobagrus fulvidraco), with rapid amplification of cDNA ends. The structure differences and the regulation effects of dietary VD3 under low phosphorus were compared among three isoforms of slc34a2 in yellow catfish. The predicted Slc34a2a2 and Slc34a2b proteins match 65 % and 53.8 % sequence identity, with Slc34a2a1, respectively. The membrane-spanning domains were different among these three isoforms. Intestinal Slc34a2a1 and Slc34a2a2 proteins had eight and eleven transmembrane domains, while renal Slc34a2b protein had nine. The tissue distribution study showed that same as slc34a2a1, slc34a2a2 mRNA was mainly distributed in intestine and slc34a2b mRNA in kidney. The effect of vitamin D3 (VD3) level on slc34a2 subfamily expression under low-phosphate conditions, induced by the addition of 0 (VD0), 324 (VD1), 1243 (VD2), 3621 (VD3), 8040 (VD4), or 22700 (VD5) IU VD3/kg feed, was assessed by qPCR. The dose-responsive expression of intestinal slc34a2a2 and high expression of intestinal slc34a2a2 in VD5 together with peak expression of kidney slc34a2b in VD3 coincided with the accumulation of body phosphate content. These data suggested that appropriate level of dietary VD3 up-regulated slc34a2a1, slc34a2a2, and slc34a2b mRNA levels, which increased phosphate retention. In conclusion, the current study provided another possible approach to improve dietary phosphate utilization by adding appropriate level of VD3 to a low-phosphate diet to regulate intestinal and renal slc34a2 gene expression and thus minimize the excretion of phosphorus in yellow catfish.

  13. Recombinant Measles AIK-C Vaccine Strain Expressing the prM-E Antigen of Japanese Encephalitis Virus.

    PubMed

    Higuchi, Akira; Toriniwa, Hiroko; Komiya, Tomoyoshi; Nakayama, Tetsuo

    2016-01-01

    An inactivated Japanese encephalitis virus (JEV) vaccine, which induces neutralizing antibodies, has been used for many years in Japan. In the present study, the JEV prM-E protein gene was cloned, inserted at the P/M junction of measles AIK-C cDNA, and an infectious virus was recovered. The JEV E protein was expressed in B95a cells infected with the recombinant virus. Cotton rats were inoculated with recombinant virus. Measles PA antibodies were detected three weeks after immunization. Neutralizing antibodies against JEV developed one week after inoculation, and EIA antibodies were detected three weeks after immunization. The measles AIK-C-based recombinant virus simultaneously induced measles and JEV immune responses, and may be a candidate for infant vaccines. Therefore, the present strategy of recombinant viruses based on a measles vaccine vector would be applicable to the platform for vaccine development.

  14. Structure and characterization of AAT-1 isoforms.

    PubMed

    Matsuda, Eiko; Ishizaki, Ray; Taira, Takahiro; Iguchi-Ariga, Sanae M M; Ariga, Hiroyoshi

    2005-05-01

    A novel protein, AAT-1, was identified as a AMY-1-binding protein and three splicing variants of AAT-1, AAT-1alpha, -beta and -gamma were identified. The function of AAT-1 is thought to be related to spermatogenesis. In this study, we further identified other splicing isoforms of AAT-1, AAT-1L, AAT-1M and AAT-1S, consisting of 767, 603 and 252 amino acids, respectively. These isoforms were found to use a promoter different from that used by AAT-1alpha, -beta and -gamma in the aat-1 gene, which contains 20 exons. Only 60 amino acids in the C-terminal portion of AAT-1 derived from exons 15-17 are common among AAT-1L, AAT-1M, AAT-1S and AAT-1alpha. While AAT-1alpha is specifically expressed in the testis, AAT-1L, AAT-1M, AAT-1S were found to be differentially expressed in human tissues. All of the isoforms of AAT-1 were found to bind to and colocalized with AMY-1 in human cells. While AAT-1L and AAT-1M were found to be localized diffusely in the cytoplasm, AAT-1S, like AAT-1alpha, was found to be localized in the mitochondria-like structure, suggesting different roles of AAT-1 isoforms in cells.

  15. Progesterone (PR), oestrogen (ER-α and ER-β) and oxytocin (OTR) gene expression in the oviduct and uterus of pregnant and non-pregnant bitches.

    PubMed

    Derussi, A A P; de Souza, R W A; Volpato, R; Guaitolini, C R F; Ackermann, C L; Taffarel, M O; Cardoso, G S; Dal-Pai-Silva, M; Lopes, M D

    2012-12-01

    The aim was to assess hormone receptor gene expression in the oviduct and uterus during canine pregnancy. Nineteen pregnant bitches divided into four groups were ovariohysterectomized (OVH) at either day 8, 12, 21 or 60 of pregnancy, and five non-pregnant females underwent OVH 12 days after the pre-ovulatory Luteinizant Hormone (LH) surge and served as controls. RT-qPCR for progesterone (PR), oestrogen (ER-α and ER-β) and oxytocin (OTR) receptors was performed on the oviduct and uterine tissue. The mRNA PR expression in the uterus during early stages of pregnancy and the luteal phase was higher than at other times. The mRNA ER-β expression in the oviduct during early pregnancy was less than in non-pregnant bitches. In the uterus, the mRNA ER-β expression was higher in the initial stages of pregnancy. The ER-α expression was higher in the oviduct and uterus in advanced stages of pregnancy. The mRNA OTR expression in the oviduct was lower than in the uterus in control group. The expression of this receptor in oviduct and the uterus was higher in the final stages of pregnancy, when compared with other phases. These data suggested that the serum progesterone concentrations probably exert a direct control on the PR and ER (α and β) expression and indirectly on OTR expression in the bitch oviduct and uterus.

  16. A novel PR10 promoter from Erianthus arundinaceus directs high constitutive transgene expression and is enhanced upon wounding in heterologous plant systems.

    PubMed

    Chakravarthi, M; Syamaladevi, Divya P; Harunipriya, P; Augustine, Sruthy Maria; Subramonian, N

    2016-01-01

    In genetic engineering, inducible promoters play an important role as the expression of genes driven by them can be turned on or off under situations like biotic or abiotic factors. There are few reports on inducible promoters that can be employed in the development of transgenic plants, particularly in sugarcane. In the present study, four wound inducible genes (Chitinase, PR1A, PR10 and HRGP) were selected and were amplified from Erianthus arundinaceus, a distant relative of sugarcane. In order to determine the gene that is highly induced upon wounding, RT-qPCR was performed, which showed that PR10 gene expression was instantaneous and higher upon wounding when compared to the other three genes. Using the random amplification of genomic ends technique, a 592 bp promoter sequence was obtained and in silico analysis of the upstream regulatory region revealed a 469 bp promoter and 123 bp of 5' untranslated region (UTR). Functional analyses of the promoter sequence (with and without 5' UTR) in tobacco, rice and sugarcane using β-glucuronidase (GUS) as the reporter gene revealed the constitutive and inducible nature of the PR10 promoter. Our studies have demonstrated that the PR10 promoter, though highly constitutive, was quickly induced upon wounding as well as on treatment with abscisic acid and methyl jasmonate hormones. This is the first report on the isolation and characterization of a PR10 promoter from a wild grass and is expected to have application for development of transgenic plants.

  17. Fusion of TTYH1 with the C19MC microRNA cluster drives expression of a brain-specific DNMT3B isoform in the embryonal brain tumor ETMR.

    PubMed

    Kleinman, Claudia L; Gerges, Noha; Papillon-Cavanagh, Simon; Sin-Chan, Patrick; Pramatarova, Albena; Quang, Dong-Anh Khuong; Adoue, Véronique; Busche, Stephan; Caron, Maxime; Djambazian, Haig; Bemmo, Amandine; Fontebasso, Adam M; Spence, Tara; Schwartzentruber, Jeremy; Albrecht, Steffen; Hauser, Peter; Garami, Miklos; Klekner, Almos; Bognar, Laszlo; Montes, Jose-Luis; Staffa, Alfredo; Montpetit, Alexandre; Berube, Pierre; Zakrzewska, Magdalena; Zakrzewski, Krzysztof; Liberski, Pawel P; Dong, Zhifeng; Siegel, Peter M; Duchaine, Thomas; Perotti, Christian; Fleming, Adam; Faury, Damien; Remke, Marc; Gallo, Marco; Dirks, Peter; Taylor, Michael D; Sladek, Robert; Pastinen, Tomi; Chan, Jennifer A; Huang, Annie; Majewski, Jacek; Jabado, Nada

    2014-01-01

    Embryonal tumors with multilayered rosettes (ETMRs) are rare, deadly pediatric brain tumors characterized by high-level amplification of the microRNA cluster C19MC. We performed integrated genetic and epigenetic analyses of 12 ETMR samples and identified, in all cases, C19MC fusions to TTYH1 driving expression of the microRNAs. ETMR tumors, cell lines and xenografts showed a specific DNA methylation pattern distinct from those of other tumors and normal tissues. We detected extreme overexpression of a previously uncharacterized isoform of DNMT3B originating at an alternative promoter that is active only in the first weeks of neural tube development. Transcriptional and immunohistochemical analyses suggest that C19MC-dependent DNMT3B deregulation is mediated by RBL2, a known repressor of DNMT3B. Transfection with individual C19MC microRNAs resulted in DNMT3B upregulation and RBL2 downregulation in cultured cells. Our data suggest a potential oncogenic re-engagement of an early developmental program in ETMR via epigenetic alteration mediated by an embryonic, brain-specific DNMT3B isoform.

  18. T cell receptor complexes containing Fc epsilon RI gamma homodimers in lieu of CD3 zeta and CD3 eta components: a novel isoform expressed on large granular lymphocytes

    PubMed Central

    1992-01-01

    CD3 zeta and CD3 eta form disulfide-linked homo- or heterodimers important in targeting partially assembled Ti alpha-beta/CD3 gamma delta epsilon T cell receptor (TCR) complexes to the cell surface and transducing stimulatory signals after antigen recognition. Here we identify a new TCR isoform expressed on splenic CD2+, CD3/Ti alpha- beta+, CD4-, CD8-, CD16+, NK1.1+ mouse large granular lymphocytes (LGL), which are devoid of CD3 zeta and CD3 eta proteins. The TCRs of this subset contain homodimers of the gamma subunit of the high affinity receptor for IgE (Fc epsilon RI gamma) in lieu of CD3 zeta and/or CD3 eta proteins. The LGL display natural killer-like activity and are cytotoxic for B cell hybridomas producing anti-CD3 epsilon and anti-CD16 monoclonal antibodies, demonstrating the signaling capacity of both TCR and CD16 in this cell type. These findings provide evidence for an additional level of complexity of TCR signal transduction isoforms in naturally occurring T cell subsets. PMID:1530959

  19. Survivin isoform Delta Ex3 regulates tumor spheroid formation.

    PubMed

    Espinosa, Magali; Ceballos-Cancino, Gisela; Callaghan, Richard; Maldonado, Vilma; Patiño, Nelly; Ruíz, Víctor; Meléndez-Zajgla, Jorge

    2012-05-01

    Survivin is an important member of the Inhibitor of Apoptosis Proteins (IAPs) family and has essential roles in apoptosis and cell cycle progression. This gene is commonly upregulated in human cancer and provides an exciting diagnostic and therapeutic target. Survivin is expressed as several isoforms that are generated by alternative splicing, and some of these present antagonistic activities. Currently, information regarding the regulation of these isoforms is lacking. In this study, we sought to analyze survivin Delta Ex3 expression in a three-dimensional model of avascular tumors and its overexpression effects in processes such as proliferation, clonogenicity and apoptosis. We found a positive correlation between spheroid growth and survivin Delta Ex3 expression during the exponential phase. We demonstrated that this isoform not only decreased apoptosis but also inhibited tumor spheroid formation by decreasing proliferation and clonogenic survival. These results point toward a dual and antagonistic effect of this spliced survivin isoform in cancer development.

  20. cDNA cloning, expression, and mutagenesis of a PR-10 protein SPE-16 from the seeds of Pachyrrhizus erosus.

    PubMed

    Wu, Fang; Yan, Ming; Li, Yikun; Chang, Shaojie; Song, Xiaomin; Zhou, Zhaocai; Gong, Weimin

    2003-12-19

    SPE-16 is a new 16kDa protein that has been purified from the seeds of Pachyrrhizus erosus. It's N-terminal amino acid sequence shows significant sequence homology to pathogenesis-related class 10 proteins. cDNA encoding 150 amino acids was cloned by RT-PCR and the gene sequence proved SPE-16 to be a new member of PR-10 family. The cDNA was cloned into pET15b plasmid and expressed in Escherichia coli. The bacterially expressed SPE-16 also demonstrated ribonuclease-like activity in vitro. Site-directed mutation of three conserved amino acids E95A, E147A, Y150A, and a P-loop truncated form were constructed and their different effects on ribonuclease activities were observed. SPE-16 is also able to bind the fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS) in the native state. The ANS anion is a much-utilized "hydrophobic probe" for proteins. This binding activity indicated another biological function of SPE-16.

  1. Tunable protein synthesis by transcript isoforms in human cells

    PubMed Central

    Floor, Stephen N; Doudna, Jennifer A

    2016-01-01

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

  2. Prion protein expression in bovine podocytes and extraglomerular mesangial cells.

    PubMed

    Amselgruber, W M; Steffl, M; Didier, A; Märtlbauer, E; Pfaff, E; Büttner, M

    2006-06-01

    The cellular form of the prion protein (PrP(c)) is thought to be a substrate for an abnormal isoform of the prion protein (PrP(sc)). One emerging hypothesis is that the proposed conversion phenomenon takes place at the site at which the infectious agent meets PrP(c). PrP(c) is abundant in the central nervous system, but little is known about the cell-type-specific distribution of PrP(c) in non-neuronal tissues of cattle. We have studied whether PrP(c), a protein found predominantly in neurons, also exists in bovine podocytes, since neurons and podocytes share a large number of similarities. We have therefore examined the expression of PrP(c) by immunohistochemistry, reverse transcription/polymerase chain reaction and enzyme-linked immunosorbent analysis. Immunostained serial sections and specific antibodies against PrP(c) have revealed that PrP(c) is selectively localized in podocytes and is particularly strongly expressed in extraglomerular mesangial cells but not in endothelial or intraglomerular mesangial cells. The selective expression of PrP(c) in podocytes is of special importance, as it suggests that these cells represent possible targets for peripheral infection with prions and demonstrates that PrP(c) can be added to the list of neuronal factors expressed in mammalian podocytes.

  3. The C-Terminal Domain of Nrf1 Negatively Regulates the Full-Length CNC-bZIP Factor and Its Shorter Isoform LCR-F1/Nrf1β; Both Are Also Inhibited by the Small Dominant-Negative Nrf1γ/δ Isoforms that Down-Regulate ARE-Battery Gene Expression

    PubMed Central

    Zhang, Yiguo; Qiu, Lu; Li, Shaojun; Xiang, Yuancai; Chen, Jiayu; Ren, Yonggang

    2014-01-01

    The C-terminal domain (CTD, aa 686–741) of nuclear factor-erythroid 2 p45-related factor 1 (Nrf1) shares 53% amino acid sequence identity with the equivalent Neh3 domain of Nrf2, a homologous transcription factor. The Neh3 positively regulates Nrf2, but whether the Neh3-like (Neh3L) CTD of Nrf1 has a similar role in regulating Nrf1-target gene expression is unknown. Herein, we report that CTD negatively regulates the full-length Nrf1 (i.e. 120-kDa glycoprotein and 95-kDa deglycoprotein) and its shorter isoform LCR-F1/Nrf1β (55-kDa). Attachment of its CTD-adjoining 112-aa to the C-terminus of Nrf2 yields the chimaeric Nrf2-C112Nrf1 factor with a markedly decreased activity. Live-cell imaging of GFP-CTD reveals that the extra-nuclear portion of the fusion protein is allowed to associate with the endoplasmic reticulum (ER) membrane through the amphipathic Neh3L region of Nrf1 and its basic c-tail. Thus removal of either the entire CTD or the essential Neh3L portion within CTD from Nrf1, LCR-F1/Nrf1β and Nrf2-C112Nrf1, results in an increase in their transcriptional ability to regulate antioxidant response element (ARE)-driven reporter genes. Further examinations unravel that two smaller isoforms, 36-kDa Nrf1γ and 25-kDa Nrf1δ, act as dominant-negative inhibitors to compete against Nrf1, LCR-F1/Nrf1β and Nrf2. Relative to Nrf1, LCR-F1/Nrf1β is a weak activator, that is positively regulated by its Asn/Ser/Thr-rich (NST) domain and acidic domain 2 (AD2). Like AD1 of Nrf1, both AD2 and NST domain of LCR-F1/Nrf1β fused within two different chimaeric contexts to yield Gal4D:Nrf1β607 and Nrf1β:C270Nrf2, positively regulate their transactivation activity of cognate Gal4- and Nrf2-target reporter genes. More importantly, differential expression of endogenous ARE-battery genes is attributable to up-regulation by Nrf1 and LCR-F1/Nrf1β and down-regulation by Nrf1γ and Nrf1δ. PMID:25290918

  4. The C-terminal domain of Nrf1 negatively regulates the full-length CNC-bZIP factor and its shorter isoform LCR-F1/Nrf1β; both are also inhibited by the small dominant-negative Nrf1γ/δ isoforms that down-regulate ARE-battery gene expression.

    PubMed

    Zhang, Yiguo; Qiu, Lu; Li, Shaojun; Xiang, Yuancai; Chen, Jiayu; Ren, Yonggang

    2014-01-01

    The C-terminal domain (CTD, aa 686-741) of nuclear factor-erythroid 2 p45-related factor 1 (Nrf1) shares 53% amino acid sequence identity with the equivalent Neh3 domain of Nrf2, a homologous transcription factor. The Neh3 positively regulates Nrf2, but whether the Neh3-like (Neh3L) CTD of Nrf1 has a similar role in regulating Nrf1-target gene expression is unknown. Herein, we report that CTD negatively regulates the full-length Nrf1 (i.e. 120-kDa glycoprotein and 95-kDa deglycoprotein) and its shorter isoform LCR-F1/Nrf1β (55-kDa). Attachment of its CTD-adjoining 112-aa to the C-terminus of Nrf2 yields the chimaeric Nrf2-C112Nrf1 factor with a markedly decreased activity. Live-cell imaging of GFP-CTD reveals that the extra-nuclear portion of the fusion protein is allowed to associate with the endoplasmic reticulum (ER) membrane through the amphipathic Neh3L region of Nrf1 and its basic c-tail. Thus removal of either the entire CTD or the essential Neh3L portion within CTD from Nrf1, LCR-F1/Nrf1β and Nrf2-C112Nrf1, results in an increase in their transcriptional ability to regulate antioxidant response element (ARE)-driven reporter genes. Further examinations unravel that two smaller isoforms, 36-kDa Nrf1γ and 25-kDa Nrf1δ, act as dominant-negative inhibitors to compete against Nrf1, LCR-F1/Nrf1β and Nrf2. Relative to Nrf1, LCR-F1/Nrf1β is a weak activator, that is positively regulated by its Asn/Ser/Thr-rich (NST) domain and acidic domain 2 (AD2). Like AD1 of Nrf1, both AD2 and NST domain of LCR-F1/Nrf1β fused within two different chimaeric contexts to yield Gal4D:Nrf1β607 and Nrf1β:C270Nrf2, positively regulate their transactivation activity of cognate Gal4- and Nrf2-target reporter genes. More importantly, differential expression of endogenous ARE-battery genes is attributable to up-regulation by Nrf1 and LCR-F1/Nrf1β and down-regulation by Nrf1γ and Nrf1δ.

  5. Physiological TLR5 expression in the intestine is regulated by differential DNA binding of Sp1/Sp3 through simultaneous Sp1 dephosphorylation and Sp3 phosphorylation by two different PKC isoforms.

    PubMed

    Thakur, Bhupesh Kumar; Dasgupta, Nirmalya; Ta, Atri; Das, Santasabuj

    2016-07-08

    Toll-like receptor 5 (TLR5) expression in the intestinal epithelial cells (IECs) is critical to maintain health, as underscored by multiple intestinal and extra-intestinal diseases in mice genetically engineered for IEC-specific TLR5 knockout. A gradient of expression exists in the colonic epithelial cells from the cecum to the distal colon. Intriguingly, an identical gradient for the dietary metabolite, butyrate also exists in the luminal contents. However, both being critical for intestinal homeostasis and immune response, no studies examined the role of butyrate in the regulation of TLR5 expression. We showed that butyrate transcriptionally upregulates TLR5 in the IECs and augments flagellin-induced immune responses. Both basal and butyrate-induced transcription is regulated by differential binding of Sp-family transcription factors to the GC-box sequences over the TLR5 promoter. Butyrate activates two different protein kinase C isoforms to dephosphorylate/acetylate Sp1 by serine/threonine phosphatases and phosphorylate Sp3 by ERK-MAPK, respectively. This resulted in Sp1 displacement from the promoter and binding of Sp3 to it, leading to p300 recruitment and histone acetylation, activating transcription. This is the first study addressing the mechanisms of physiological TLR5 expression in the intestine. Additionally, a novel insight is gained into Sp1/Sp3-mediated gene regulation that may apply to other genes.

  6. Physiological TLR5 expression in the intestine is regulated by differential DNA binding of Sp1/Sp3 through simultaneous Sp1 dephosphorylation and Sp3 phosphorylation by two different PKC isoforms

    PubMed Central

    Thakur, Bhupesh Kumar; Dasgupta, Nirmalya; Ta, Atri; Das, Santasabuj

    2016-01-01

    Toll-like receptor 5 (TLR5) expression in the intestinal epithelial cells (IECs) is critical to maintain health, as underscored by multiple intestinal and extra-intestinal diseases in mice genetically engineered for IEC-specific TLR5 knockout. A gradient of expression exists in the colonic epithelial cells from the cecum to the distal colon. Intriguingly, an identical gradient for the dietary metabolite, butyrate also exists in the luminal contents. However, both being critical for intestinal homeostasis and immune response, no studies examined the role of butyrate in the regulation of TLR5 expression. We showed that butyrate transcriptionally upregulates TLR5 in the IECs and augments flagellin-induced immune responses. Both basal and butyrate-induced transcription is regulated by differential binding of Sp-family transcription factors to the GC-box sequences over the TLR5 promoter. Butyrate activates two different protein kinase C isoforms to dephosphorylate/acetylate Sp1 by serine/threonine phosphatases and phosphorylate Sp3 by ERK-MAPK, respectively. This resulted in Sp1 displacement from the promoter and binding of Sp3 to it, leading to p300 recruitment and histone acetylation, activating transcription. This is the first study addressing the mechanisms of physiological TLR5 expression in the intestine. Additionally, a novel insight is gained into Sp1/Sp3-mediated gene regulation that may apply to other genes. PMID:27060138

  7. Characteristic expression of twelve rice PR1 family genes in response to pathogen infection, wounding, and defense-related signal compounds (121/180)

    PubMed Central

    Iwai, Takayoshi; Seo, Shigemi; Yanagawa, Yuki; Kawahigasi, Hiroyuki; Hirose, Sakino; Ohkawa, Yasunobu; Ohashi, Yuko

    2008-01-01

    Pathogenesis-related (PR) proteins have been used as markers of plant defense responses, and are classified into 17 families. However, precise information on the majority members in specific PR families is still limited. We were interested in the individual characteristics of rice PR1 family genes, and selected 12 putatively active genes using rice genome databases for expressed genes. All were upregulated upon compatible and/or incompatible rice-blast fungus interactions; three were upregulated in the early infection period and four in the late infection period. Upon compatible rice–bacterial blight interaction, four genes were upregulated, six were not affected, and one was downregulated. These results are in striking contrast to those among 22 ArabidopsisPR1 genes where only one gene was pathogen-inducible. The responses of individual genes to salicylic acid, jasmonic acid, and ethylene induced defense signaling pathways in rice are likely to be different from those in dicot plants. Transcript levels in healthy leaves, roots, and flowers varied according to each gene. Analysis of the partially overlapping expression patterns of rice PR1 genes in healthy tissues and in response to pathogens and other stresses would be useful to understand their possible functions and for use as characteristic markers for defense-related studies in rice. PMID:18247056

  8. Absolute quantitation of protein posttranslational modification isoform.

    PubMed

    Yang, Zhu; Li, Ning

    2015-01-01

    -site-independent peptides in the total cellular protein and their peptide yields. The PTM occupancy determination is achieved by measuring the absolute amounts of both PTM and non-PTM peptides from the highly purified protein sample expressed in transgenic organisms or directly isolated from an organism using affinity purification. The absolute amount of each PTM isoform in the total cellular protein extract is finally calculated from these two variables. Following this approach, the ion intensities given by mass spectrometers are used to calculated the peptide amounts, from which the amounts of protein isoforms are then deduced. In this chapter, we describe the principles underlying the experimental design and procedures used in AQUIP method. This quantitation method basically employs stable isotope-labeled peptide standards and affinity purification from a tagged recombinant protein of interest. Other quantitation strategies and purification techniques related to this method are also discussed.

  9. Voltage-Gated Na+ Channel Isoforms and Their mRNA Expression Levels and Protein Abundance in Three Electric Organs and the Skeletal Muscle of the Electric Eel Electrophorus electricus

    PubMed Central

    Hiong, Kum C.; Boo, Mel V.; Wong, Wai P.; Chew, Shit F.

    2016-01-01

    This study aimed to obtain the coding cDNA sequences of voltage-gated Na+ channel (scn) α-subunit (scna) and β-subunit (scnb) isoforms from, and to quantify their transcript levels in, the main electric organ (EO), Hunter’s EO, Sach’s EO and the skeletal muscle (SM) of the electric eel, Electrophorus electricus, which can generate both high and low voltage electric organ discharges (EODs). The full coding sequences of two scna (scn4aa and scn4ab) and three scnb (scn1b, scn2b and scn4b) were identified for the first time (except scn4aa) in E. electricus. In adult fish, the scn4aa transcript level was the highest in the main EO and the lowest in the Sach’s EO, indicating that it might play an important role in generating high voltage EODs. For scn4ab/Scn4ab, the transcript and protein levels were unexpectedly high in the EOs, with expression levels in the main EO and the Hunter’s EO comparable to those of scn4aa. As the key domains affecting the properties of the channel were mostly conserved between Scn4aa and Scn4ab, Scn4ab might play a role in electrogenesis. Concerning scnb, the transcript level of scn4b was much higher than those of scn1b and scn2b in the EOs and the SM. While the transcript level of scn4b was the highest in the main EO, protein abundance of Scn4b was the highest in the SM. Taken together, it is unlikely that Scna could function independently to generate EODs in the EOs as previously suggested. It is probable that different combinations of Scn4aa/Scn4ab and various Scnb isoforms in the three EOs account for the differences in EODs produced in E. electricus. In general, the transcript levels of various scn isoforms in the EOs and the SM were much higher in adult than in juvenile, and the three EOs of the juvenile fish could be functionally indistinct. PMID:27907137

  10. Progesterone receptor isoform functions in normal breast development and breast cancer.

    PubMed

    Kariagina, Anastasia; Aupperlee, Mark D; Haslam, Sandra Z

    2008-01-01

    Progesterone acting through two isoforms of the progesterone receptor (PR), PRA and PRB, regulates proliferation and differentiation in the normal mammary gland in mouse, rat, and human. Progesterone and PR have also been implicated in the etiology and pathogenesis of human breast cancer. The focus of this review is recent advances in understanding the role of the PR isoform-specific functions in the normal breast and in breast cancer. Also discussed is information obtained from rodent studies and their relevance to our understanding of the role of progestins in breast cancer etiology.

  11. Brain delivery of AAV9 expressing an anti-PrP monovalent antibody delays prion disease in mice.

    PubMed

    Moda, Fabio; Vimercati, Chiara; Campagnani, Ilaria; Ruggerone, Margherita; Giaccone, Giorgio; Morbin, Michela; Zentilin, Lorena; Giacca, Mauro; Zucca, Ileana; Legname, Giuseppe; Tagliavini, Fabrizio

    2012-01-01

    Prion diseases are caused by a conformational modification of the cellular prion protein (PrP (C)) into disease-specific forms, termed PrP (Sc), that have the ability to interact with PrP (C) promoting its conversion to PrP (Sc). In vitro studies demonstrated that anti-PrP antibodies inhibit this process. In particular, the single chain variable fragment D18 antibody (scFvD18) showed high efficiency in curing chronically prion-infected cells. This molecule binds the PrP (C) region involved in the interaction with PrP (Sc) thus halting further prion formation. These findings prompted us to test the efficiency of scFvD18 in vivo. A recombinant Adeno-Associated Viral vector serotype 9 was used to deliver scFvD18 to the brain of mice that were subsequently infected by intraperitoneal route with the mouse-adapted scrapie strain RML. We found that the treatment was safe, prolonged the incubation time of scrapie-infected animals and decreased the burden of total proteinase-resistant PrP (Sc) in the brain, suggesting that scFvD18 interferes with prion replication in vivo. This approach is relevant for designing new therapeutic strategies for prion diseases and other disorders characterized by protein misfolding.

  12. Mitotic-specific methylation of histone H4 Lys 20 follows increased PR-Set7 expression and its localization to mitotic chromosomes

    PubMed Central

    Rice, Judd C.; Nishioka, Kenichi; Sarma, Kavitha; Steward, Ruth; Reinberg, Danny; Allis, C. David

    2002-01-01

    We describe distinct patterns of histone methylation during human cell cycle progression. Histone H4 methyltransferase activity was found to be cell cycle-regulated, consistent with increased H4 Lys 20 methylation at mitosis. This increase closely followed the cell cycle-regulated expression of the H4 Lys 20 methyltransferase, PR-Set7. Localization of PR-Set7 to mitotic chromosomes and subsequent increase in H4 Lys 20 methylation were inversely correlated to transient H4 Lys 16 acetylation in early S-phase. These data suggest that H4 Lys 20 methylation by PR-Set7 during mitosis acts to antagonize H4 Lys 16 acetylation and to establish a mechanism by which this mark is epigenetically transmitted. PMID:12208845

  13. Effects of Ethanol on the Expression Level of Various BDNF mRNA Isoforms and Their Encoded Protein in the Hippocampus of Adult and Embryonic Rats

    PubMed Central

    Shojaei, Shahla; Ghavami, Saeid; Panjehshahin, Mohammad Reza; Owji, Ali Akbar

    2015-01-01

    We aimed to compare the effects of oral ethanol (Eth) alone or combined with the phytoestrogen resveratrol (Rsv) on the expression of various brain-derived neurotrophic factor (BDNF) transcripts and the encoded protein pro-BDNF in the hippocampus of pregnant and embryonic rats. A low (0.25 g/kg body weight (BW)/day) dose of Eth produced an increase in the expression of BDNF exons I, III and IV and a decrease in that of the exon IX in embryos, but failed to affect BDNF transcript and pro-BDNF protein expression in adults. However, co-administration of Eth 0.25 g/kg·BW/day and Rsv led to increased expression of BDNF exons I, III and IV and to a small but significant increase in the level of pro-BDNF protein in maternal rats. A high (2.5 g/kg·BW/day) dose of Eth increased the expression of BDNF exons III and IV in embryos, but it decreased the expression of exon IX containing BDNF mRNAs in the maternal rats. While the high dose of Eth alone reduced the level of pro-BDNF in adults, it failed to change the levels of pro-BDNF in embryos. Eth differentially affects the expression pattern of BDNF transcripts and levels of pro-BDNF in the hippocampus of both adult and embryonic rats. PMID:26703578

  14. Differential activities of glucocorticoid-induced leucine zipper protein isoforms.

    PubMed

    Soundararajan, Rama; Wang, Jian; Melters, Daniël; Pearce, David

    2007-12-14

    Glucocorticoid-induced leucine zipper protein (GILZ) is expressed in both epithelial and immune tissues and modulates a variety of cellular functions, including proliferation and epithelial sodium channel (ENaC) activity. A number of reports have described various GILZ activities, focusing on a single isoform with molecular mass of approximately 17 kDa, now termed GILZ1. In GILZ immunoblots using a newly developed antiserum, we detected multiple species in extracts from cultured kidney cells. Mass spectrometric analysis revealed that one of these represented a previously uncharacterized distinct isoform of GILZ, GILZ2. Rapid amplification of cDNA ends was used to clone cDNAs corresponding to four isoforms, which, in addition to GILZ1 and GILZ2, included new isoforms GILZ3 and GILZ4. Heterologous expression of these four GILZ isoforms in cultured cells revealed striking functional differences. Notably, GILZ1 was the only isoform that significantly stimulated ENaC-mediated Na+ current in a kidney collecting duct cell line, although GILZ2 and GILZ3 also stimulated ENaC surface expression in HEK 293 cells. GILZ1 and GILZ3, and to a lesser extent GILZ2, inhibited ERK phosphorylation. Interestingly, GILZ4, which had no effect on either ENaC or ERK, potently suppressed cellular proliferation, as did GILZ1, but not GILZ2 or GILZ3. Finally, rat and mouse tissues all expressed multiple GILZ species but varied in the relative abundance of each. These data suggest that multiple GILZ isoforms are expressed in most cells and tissues and that these play distinct roles in regulating key cellular functions, including proliferation and ion transport. Furthermore, GILZ inhibition of ERK appears to play an essential role in stimulation of cell surface ENaC but not in inhibition of proliferation.

  15. Nonmuscle myosin II isoforms coassemble in living cells.

    PubMed

    Beach, Jordan R; Shao, Lin; Remmert, Kirsten; Li, Dong; Betzig, Eric; Hammer, John A

    2014-05-19

    Nonmuscle myosin II (NM II) powers myriad developmental and cellular processes, including embryogenesis, cell migration, and cytokinesis [1]. To exert its functions, monomers of NM II assemble into bipolar filaments that produce a contractile force on the actin cytoskeleton. Mammalian cells express up to three isoforms of NM II (NM IIA, IIB, and IIC), each of which possesses distinct biophysical properties and supports unique as well as redundant cellular functions [2-8]. Despite previous efforts [9-13], it remains unclear whether NM II isoforms assemble in living cells to produce mixed (heterotypic) bipolar filaments or whether filaments consist entirely of a single isoform (homotypic). We addressed this question using fluorescently tagged versions of NM IIA, IIB, and IIC, isoform-specific immunostaining of the endogenous proteins, and two-color total internal reflection fluorescence structured-illumination microscopy, or TIRF-SIM, to visualize individual myosin II bipolar filaments inside cells. We show that NM II isoforms coassemble into heterotypic filaments in a variety of settings, including various types of stress fibers, individual filaments throughout the cell, and the contractile ring. We also show that the differential distribution of NM IIA and NM IIB typically seen in confocal micrographs of well-polarized cells is reflected in the composition of individual bipolar filaments. Interestingly, this differential distribution is less pronounced in freshly spread cells, arguing for the existence of a sorting mechanism acting over time. Together, our work argues that individual NM II isoforms are potentially performing both isoform-specific and isoform-redundant functions while coassembled with other NM II isoforms.

  16. Over-expression of a cytosolic isoform of the HbCuZnSOD gene in Hevea brasiliensis changes its response to a water deficit.

    PubMed

    Leclercq, J; Martin, F; Sanier, C; Clément-Vidal, A; Fabre, D; Oliver, G; Lardet, L; Ayar, A; Peyramard, M; Montoro, P

    2012-10-01

    Hevea brasiliensis is the main commercial source of natural rubber. Reactive oxygen species (ROS) scavenging systems are involved in various biotic and abiotic stresses. Genetic engineering was undertaken to study the strengthening of plant defences by antioxidants. To that end, Hevea transgenic plant lines over-expressing a Hevea brasiliensis cytosolic HbCuZnSOD gene were successfully established and regenerated. Over-expression of the HbCuZnSOD gene was not clearly related to an increase in SOD activity in plant leaves. The impact of HbCuZnSOD gene over-expression in somatic embryogenesis and in plant development are presented and discussed. The water deficit tolerance of two HbCuZnSOD over-expressing lines was evaluated. The physiological parameters of transgenic plantlets subjected to a water deficit suggested that plants from line TS4T8An displayed lower stomatal conductance and a higher proline content. Over-expression of the HbCuZnSOD gene and activation of all ROS-scavenging enzymes also suggested that protection against ROS was more efficient in the TS4T8An transgenic line.

  17. Pfkfb (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) isoforms display a tissue-specific and dynamic expression during Xenopus laevis development.

    PubMed

    Pegoraro, Caterina; Maczkowiak, Frederique; Monsoro-Burq, Anne H

    2013-10-01

    Pfkfb (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) enzymes are bi-functional enzymes encoded by four different genes (pfkfb1, pfkfb2, pfkfb3, pfkfb4) in vertebrates. They are involved in the regulation of glycolysis: they catalyze the synthesis and the degradation of F-2,6-BP (fructose-2,6-bisphosphate), the most potent allosteric activator of phosphofructokinase 1 (Pfk1), a key glycolytic enzyme. By producing F-2,6-BP, Pfkfb enzymes allow glycolysis to proceed, while by degrading F-2,6-BP they block glycolysis. As major regulators of glycolysis, Pfkfb enzymes are involved in cancer: tumor cells have a higher glycolytic rate compared to normal cells, even in the presence of adequate oxygen levels (Warburg effect) and several cancer cell lines express elevated levels of Pfkfb enzymes. Glycolysis is also important for energy and metabolite production in proliferating cells. In embryos, however, the role of glycolysis and the expression of glycolysis regulators remain to be explored. Here, we provide a phylogenetic analysis of Pfkfb enzymes in vertebrates, and we detail the expression pattern of pfk1, pfkfb1, pfkfb2, pfkfb3, and pfkfb4 genes in Xenopus laevis embryos. We show that pfkfb transcripts expression is overlapping at blastula and gastrula stages and that from neurulation to tadpole stages, they display tissue-specific, complementary and dynamic expression patterns.

  18. Regulation of CDPK isoforms during tuber development.

    PubMed

    Raíces, Marcela; Gargantini, Pablo Rubén; Chinchilla, Delphine; Crespi, Martín; Téllez-Iñón, María Teresa; Ulloa, Rita María

    2003-07-01

    CDPK activities present during tuber development were analysed. A high CDPK activity was detected in the soluble fraction of early stolons and a lower one was detected in soluble and particulate fractions of induced stolons. The early and late CDPK activities displayed diverse specificity for in vitro substrates and different subcellular distribution. Western blot analysis revealed two CDPKs of 55 and 60 kDa that follow a precise spatial and temporal profile of expression. The 55 kDa protein was only detected in early-elongating stolons and the 60 kDa one was induced upon stolon swelling, correlating with early and late CDPK activities. A new member of the potato CDPK family, StCDPK3, was identified from a stolon cDNA library. Gene specific RT-PCR demonstrated that this gene is only expressed in early stolons, while the previously identified StCDPK1 is expressed upon stolon swelling. This expression profile suggests that StCDPK3 could correspond to the 55 kDa isoform while StCDPK1 could encode the 60 kDa isoform present in swelling stolons. StCDPK1 has myristoylation and palmitoylation consensus possibly involved in its dual intracellular localization. Transient expression studies with wild-type and mutated forms of StCDPK1 fused to GFP were used to show that subcellular localization of this isoform is controlled by myristoylation and palmitoylation. Altogether, our data suggest that sequential activation of StCDPK3 and StCDPK1 and the subcellular localisation of StCDPK1 might be critical regulatory steps of calcium signalling during potato tuber development.

  19. Molecular cloning and characterization of two novel genes from hexaploid wheat that encode double PR-1 domains coupled with a receptor-like protein kinase.

    PubMed

    Lu, Shunwen; Faris, Justin D; Edwards, Michael C

    2017-04-01

    Hexaploid wheat (Triticum aestivum L.) contains at least 23 TaPr-1 genes encoding the group 1 pathogenesis-related (PR-1) proteins as identified in our previous work. Here, we report the cloning and characterization of TaPr-1-rk1 and TaPr-1-rk2, two novel genes closely related to the wheat PR-1 family. The two TaPr-1-rk genes are located on homoeologous chromosomes 3D and 3A, respectively, and each contains a large open reading frame (7385 or 6060 bp) that is interrupted by seven introns and subjected to alternative splicing (AS) with five or six isoforms of mRNA transcripts. The deduced full-length TaPR-1-RK1 and TaPR-1-RK2 proteins (95% identity) contain two repeat PR-1 domains, the second of which is fused via a transmembrane helix to a serine/threonine kinase catalytic (STKc) domain characteristic of receptor-like protein kinases. Phylogenetic analysis indicated that the two PR-1 domains of the TaPR-1-RK proteins form sister clades with their homologues identified in other monocot plants and are well separated from stand-alone PR-1 proteins, whereas the STKc domains may have originated from cysteine-rich receptor-like kinases (CRKs). Reverse-transcriptase-PCR analysis revealed that the TaPr-1-rk genes are predominantly expressed in wheat leaves and their expression levels are elevated in response to pathogen attack, such as infection by barley stripe mosaic virus (BSMV), and also to stress conditions, most obviously, to soil salinity. This is the first report of PR-1-CRK hybrid proteins in wheat. The data may shed new insights into the function/evolutionary origin of the PR-1 family and the STKc-mediated defense/stress response pathways in plants.

  20. Differential protein expression of peroxiredoxin I and II by benzo(a)pyrene and quercetin treatment in 22Rv1 and PrEC prostate cell lines

    SciTech Connect

    Chaudhary, Amit; Pechan, Tibor; Willett, Kristine L. . E-mail: kwillett@olemiss.edu

    2007-04-15

    Mechanisms of benzo(a)pyrene (BaP)-mediated toxicity and chemopreventative potential of quercetin in prostate cancer are poorly understood. Two-dimensional gel electrophoresis was used to map the differences in protein expression in BaP (1 {mu}M)- and quercetin (5 {mu}M)-treated 22Rv1 human prostate cancer cells. As compared to DMSO, 26 proteins in BaP and 41 proteins in quercetin were found to be differentially expressed ({+-} 2-fold). Western blots confirmed that BaP increased peroxiredoxin (Prx) Prx I and decreased Prx II in 22Rv1 cells. Similar results were found in PrEC normal prostate