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Sample records for beta protein mrna

  1. Downregulation of TGF-beta1 mRNA and protein in the muscles of patients with inflammatory myopathies after treatment with high-dose intravenous immunoglobulin.

    Science.gov (United States)

    Amemiya, K; Semino-Mora, C; Granger, R P; Dalakas, M C

    2000-02-01

    We used reverse transcription-polymerase chain reaction to study the level of TGF-beta1 mRNA expression and immunocytochemistry to examine the immunoreactive TGF-beta1 in muscle biopsy specimens from five patients with dermatomyositis (DM) and five patients with inclusion body myositis (IBM) obtained before and after 3 months treatment with intravenous immunoglobulin (IVIg). At baseline, the mRNA expression of TGF-beta1 was increased up to fivefold in the muscles of DM patients compared to that of IBM patients. After IVIg, TGF-beta1 was downregulated and the TGF-beta1 mRNA decreased twofold in the muscles of patients with DM who had successfully responded to therapy, but remained unchanged in the muscles of patients with IBM who did not respond. The downregulation of TGF-beta1 in DM was associated with improvement of the muscle cytoarchitecture and reduction of the endomysial inflammation and connective tissue, suggesting that in DM the excess of TGF-beta1 may be involved in the pathogenesis of chronic inflammation, fibrosis, and accumulation of extracellular matrix proteins.

  2. Effects of long-term estrogen replacement therapy on beta-amyloid precursor protein and mRNA expression in ovariectomized rat hippocampus

    Institute of Scientific and Technical Information of China (English)

    Bo Jiang; Eryuan Liao; Liming Tan; Ruchun Dai; Zhijie Xiao

    2009-01-01

    BACKGROUND: In vitro cultures of neural stem cells have shown that estrogen can regulate beta-amyloid precursor protein (β-APP) metabolism and reduce amyloid-beta production.OBJECTIVE: To investigate the effects of long-term oral administration of compound nylestriol or low-dose 17beta-estradiol on β-APP and mRNA expression in the hippocampus of ovariectomized (OVX) rats. DESIGN, TIME AND SETTING: This randomized and controlled experiment was performed at the Animal Laboratory and Laboratory of Endocrine and Metabolic Disease, Xiangya Second Hospital of Central South University between April 2003 and May 2004.MATERIALS: According to body mass, 50 six-month-old female Sprague-Dawley rats were randomly divided into five groups (n = 10 per group): normal control, sham operation, OVX model, 17beta-estradiol (Sigma, USA), and compound nylestriol tablet (Laboratory of Endocrine and Metabolic Disease, Xiangya Second Hospital of Central South University) groups.METHODS: Rats in OVX plus 17beta-estradiol and OVX plus compound nylestriol tablet groups underwent ovariectomy. On the second day after surgery, rats were intragastrically given 17beta-estradiol (100 μg/kg), once per day or compound nylestriol tablet (0.5 mg/kg) and levonorgestrel (0.15 mg/kg) every 2 days.MAIN OUTCOME MEASURES: β-APP expression in the hippocampus of OVX rats was determined using immunohistochemistry (SABC method) and β-APP mRNA expression was analyzed by in situ hybridization. The results were quantitatively analyzed using cell counting and average optical density. RESULTS: The number and optical density of β-APP-positive neurons in every subregion of the hippocampus of OVX rats was dramatically increased compared with normal and sham operation groups following 35 weeks of administration (P < 0.05). Levels of β-APP were decreased following oral administration of compound nylestriol or 17beta-estradiol. In situ hybridization showed that long-term estrogen deficiency and oral administration

  3. Jueming Prescription reduces body weight by increasing the mRNA expressions of beta3-adrenergic receptor and uncoupling protein-2 in adipose tissue of diet-induced obese rats.

    Science.gov (United States)

    Yang, Ling; Lu, Kun; Wen, Xiu-ying; Liu, Hao; Chen, Ai-ping; Xu, Ming-wang; Zhang, Hong; Yu, Jie

    2012-10-01

    To investigate the antiobesity effect of Jueming Prescription (JMP), a Chinese herbal medicine formula, and its influence on mRNA expressions of beta3 adrenergic receptor (beta3-AR) and uncoupling protein-2 (UCP-2) in adipose tissue of diet-induced obese rats. Fifty male Sprague-Dawley rats were randomly divided into the normal control group (n =8) that was on a standard chow diet, and the obese model group (n =42) that was on a diet of high fat chow. Two weeks after the high fat diet, 29 obese rats in the obese model group were further randomly divided into 3 groups: the untreated obese model group (n =9), the metformin group (n =10, metformin 300 mg kg⁻¹ day)⁻¹, and the JMP group (n =10, JMP 4 g kg⁻¹ day⁻¹). After 8-week treatment, body weight, wet weight of visceral fat, and percentage of body fat (PBF) were measured. The levels of fasting blood glucose, serum lipids, and insulin were assessed, and insulin sensitivity index (ISI) was calculated. The adipose tissue section was stained with hematoxylin-Eosin, and the cellular diameter and quantity of adipocytes were evaluated by light microscopy. The mRNA expressions of beta3-AR and UCP-2 from the peri-renal fat tissue were determined by real-time reverse transcription polymerase chain reaction (RT-PCR). Compared with the obese model group, treatment with JMP resulted in significantly lower body weight, wet weight of visceral fat, PBF, and diameter of adipocytes, and significantly higher level of high-density lipoprotein cholesterol, ISI (all Pweight and adipocyte size; and the effect was associated with the up-regulation of beta3-AR and UCP-2 expressions in the adipose tissue and improvement of insulin sensitivity.

  4. Expression and autoregulation of transforming growth factor beta receptor mRNA in small-cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Nørgaard, P; Spang-Thomsen, M; Poulsen, H S

    1996-01-01

    In small-cell lung cancer cell lines resistance to growth inhibition by transforming growth factor (TGF)-beta 1, was previously shown to correlate with lack of TGF-beta receptor I (RI) and II (RII) proteins. To further investigate the role of these receptors, the expression of mRNA for RI, RII...

  5. An antisense oligodeoxynucleotide targeted against the type II sub. beta. regulatory subunit mRNA of protein kinase inhibits cAMP-induced differentiation in HL-60 leukemia cells without affecting phorbol ester effects

    Energy Technology Data Exchange (ETDEWEB)

    Tortora, G.; Clair, T.; Cho-Chung, Y.S. (National Institutes of Health, Bethesda, MD (USA))

    1990-01-01

    The type II{sub {beta}} regulatory subunit of cAMP-dependent protein kinase (RII{sub {beta}}) has been hypothesized to play an important role in the growth inhibition and differentiation induced by site-selective cAMP analogs in human cancer cells, but direct proof of this function has been lacking. To address this tissue, HL-60 human promyelocytic leukemia cells were exposed to RII{sub {beta}} antisense synthetic oligodeoxynucleotide, and the effects on cAMP-induced growth regulation were examined. Exposure of these cells to RII{sub {beta}} antisense oligodeoxynucleotide resulted in a decrease in cAMP analog-induced growth inhibition and differentiation without apparent effect on differentiation induced by phorbol esters. This loss in cAMP growth regulatory function correlated with a decrease in basal and induced levels of RII{sub {beta}} protein. Exposure to RII{sub {beta}} sense, RI{sub {alpha}} and RII{sub {alpha}} antisense, or irrelevant oligodeoxynucleotides had no such effect. These results show that the RII{sub {beta}} regulatory subunit of protein kinase plays a critical role in the cAMP-induced growth regulation of HL-60 leukemia cells.

  6. TGF beta 2 mRNA expression and pregnancy failure in mice.

    Science.gov (United States)

    Gorivodsky, M; Torchinsky, A; Zemliak, I; Savion, S; Fein, A; Toder, V

    1999-08-01

    We describe here a pattern of transforming growth factor (TGF) beta2 mRNA expression at the fetomaternal interface in mice with high rate of resorptions as well as its expression following maternal immunopotentiation. TGF beta 2 mRNA expression was evaluated in the uteroplacental units of mice with spontaneous (CBA/J x DBA/2J mouse combination) or cyclophosphamide (CP)-induced pregnancy loss. The effect of immunopotentiation on TGF beta 2 mRNA expression was determined in CP-treated females who underwent nonspecific immunostimulation with xenogeneic (rat) leukocytes. A quantitative analysis of TGF beta 2 mRNA level was performed using RNase protection assay. Distribution of TGF beta 2 mRNA transcripts at the fetomaternal interface was studied by in situ hybridization analysis. RNase protection analysis revealed four TGF beta 2 specific mRNA forms (330, 270, 230, and 170 bp) in the uteroplacental units of mice with either normal or decreased reproductive performance. A significant decrease (about 50%) in the level of TGF beta 2 mRNA was registered in the uteroplacental unit of mice with pregnancy loss as compared to the control mice. TGF beta 2 transcripts were abundant in the uterine epithelium and stroma. A specific hybridization signal was detected also in metrial gland cells and it was found to be substantially lower in CP-treated as compared to intact mice. In the resorbing uteroplacental unit, the expression of TGF beta 2 mRNA was completely lost in the uterine epithelium, and the number of TGF beta 2 mRNA-positive metrial gland cells was lower as compared to the control. Immunopotentiation decreased the resorption rate in mice with CP-induced pregnancy loss and caused a dramatic increase in TGF beta 2 mRNA expression: the level of TGF beta 2 mRNA was found to be higher by 2.0-3.2 fold in the uteroplacental unit of immunized as compared to nonimmunized CP-treated mice. These data suggest that distortion of TGF beta 2 expression at the fetomaternal interface

  7. mRNA and Protein Levels for GABA[subscript A][alpha]4, [alpha]5, [beta]1 and GABA[subscript B]R1 Receptors are Altered in Brains from Subjects with Autism

    Science.gov (United States)

    Fatemi, S. Hossein; Reutiman, Teri J.; Folsom, Timothy D.; Rooney, Robert J.; Patel, Diven H.; Thuras, Paul D.

    2010-01-01

    We have shown altered expression of gamma-aminobutyric acid A (GABA[subscript A]) and gamma-aminobutyric acid B (GABA[subscript B]) receptors in the brains of subjects with autism. In the current study, we sought to verify our western blotting data for GABBR1 via qRT-PCR and to expand our previous work to measure mRNA and protein levels of 3…

  8. mRNA and Protein Levels for GABA[subscript A][alpha]4, [alpha]5, [beta]1 and GABA[subscript B]R1 Receptors are Altered in Brains from Subjects with Autism

    Science.gov (United States)

    Fatemi, S. Hossein; Reutiman, Teri J.; Folsom, Timothy D.; Rooney, Robert J.; Patel, Diven H.; Thuras, Paul D.

    2010-01-01

    We have shown altered expression of gamma-aminobutyric acid A (GABA[subscript A]) and gamma-aminobutyric acid B (GABA[subscript B]) receptors in the brains of subjects with autism. In the current study, we sought to verify our western blotting data for GABBR1 via qRT-PCR and to expand our previous work to measure mRNA and protein levels of 3…

  9. Gallium nitrate regulates rat osteoblast expression of osteocalcin protein and mRNA levels.

    Science.gov (United States)

    Guidon, P T; Salvatori, R; Bockman, R S

    1993-01-01

    Gallium nitrate, a group IIIa metal salt, has been found to be clinically effective for the treatment of accelerated bone resorption in cancer-related hypercalcemia and Paget's disease. Here we report the effects of gallium nitrate on osteocalcin mRNA and protein levels on the rat osteoblast-like cell line ROS 17/2.8. Gallium nitrate reduced both constitutive and vitamin D3-stimulated osteocalcin protein levels in culture medium by one-half and osteocalcin mRNA levels to one-third to one-tenth of control. Gallium nitrate also inhibited vitamin D3 stimulation of osteocalcin and osteopontin mRNA levels but did not affect constitutive osteopontin mRNA levels. Among several different metals examined, gallium was unique in its ability to reduce osteocalcin mRNA levels without decreasing levels of other mRNAs synthesized by ROS 17/2.8 cells. The effects of gallium nitrate on osteocalcin mRNA and protein synthesis mimic those seen when ROS 17/2.8 cells are exposed to transforming growth factor beta 1 (TGF beta 1); however, TGF-beta 1 was not detected in gallium nitrate-treated ROS 17/2.8 cell media. Use of the RNA polymerase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole demonstrated that gallium nitrate did not alter the stability of osteocalcin mRNA. Transient transfection assays using the rat osteocalcin promoter linked to the bacterial reporter gene chloramphenicol acetyltransferase indicated that gallium nitrate blocked reporter gene expression stimulated by the osteocalcin promoter. This is the first reported effect of gallium nitrate on isolated osteoblast cells.

  10. mRNA expression of iron regulatory genes in beta-thalassemia intermedia and beta-thalassemia major mouse models.

    Science.gov (United States)

    Weizer-Stern, Orly; Adamsky, Konstantin; Amariglio, Ninette; Rachmilewitz, Eliezer; Breda, Laura; Rivella, Stefano; Rechavi, Gideon

    2006-07-01

    beta-Thalassemia is an inherited anemia in which synthesis of the hemoglobin beta-chain is decreased. The excess unmatched alpha-globin chains accumulate in the growing erythroid precursors, causing their premature death (ineffective erythropoiesis). Clinical features of beta-thalassemia include variably severe anemia and iron accumulation due to increased intestinal iron absorption. The most anemic patients require regular blood transfusions, which exacerbate their iron overload and result in damage to vital organs. The hepatic peptide hepcidin, a key regulator of iron metabolism in mammals, was recently found to be low in the urine of beta-thalassemia patients, compared with healthy controls, despite their iron overload. In our work, we measured by RQ-PCR the liver mRNA expression of hepcidin and other iron regulatory genes in beta-thalassemia major mouse model (C57Bl/6 Hbb(th3/th3)), and compared it with beta-thalassemia intermedia mouse model (C57Bl/6 Hbb(th3/+)) and control mice. We found decreased expression of hepcidin and TfR2 and increased expression of TfR1 and NGAL in the beta-thalassemia mouse models, compared with the control mice. Significant down-regulation of hepcidin expression in beta-thalassemia major, despite iron overload, might explain the increased iron absorption typically observed in thalassemia.

  11. When proteome meets genome: the alpha helix and the beta strand of proteins are eschewed by mRNA splice junctions and may define the minimal indivisible modules of protein architecture

    Indian Academy of Sciences (India)

    Sailen Barik

    2004-09-01

    The significance of the intron-exon structure of genes is a mystery. As eukaryotic proteins are made up of modular functional domains, each exon was suspected to encode some form of module; however, the definition of a module remained vague. Comparison of pre-mRNA splice junctions with the three-dimensional architecture of its protein product from different eukaryotes revealed that the junctions were far less likely to occur inside the -helices and -strands of proteins than within the more flexible linker regions (‘turns’ and ‘loops’) connecting them. The splice junctions were equally distributed in the different types of linkers and throughout the linker sequence, although a slight preference for the central region of the linker was observed. The avoidance of the -helix and the -strand by splice junctions suggests the existence of a selection pressure against their disruption, perhaps underscoring the investment made by nature in building these intricate secondary structures. A corollary is that the helix and the strand are the smallest integral architectural units of a protein and represent the minimal modules in the evolution of protein structure. These results should find use in comparative genomics, designing of cloning strategies, and in the mutual verification of genome sequences with protein structures.

  12. Increased IL-10 mRNA and IL-23 mRNA expression in multiple sclerosis: interferon-beta treatment increases IL-10 mRNA expression while reducing IL-23 mRNA expression

    DEFF Research Database (Denmark)

    Krakauer, M.; Sorensen, P.; Khademi, M.

    2008-01-01

    of the regulatory cytokine IL-10. The elevated IL-23 mRNA levels in MS patients are noteworthy in view of the newly discovered IL-23-driven Th17 T-cell subset, which is crucial in animal models of MS. Since IFN-beta therapy resulted in decreased IL-23 mRNA levels, the Th17 axis could be another target of IFN...

  13. GLUT3 protein and mRNA in autopsy muscle specimens

    Science.gov (United States)

    Stuart, C. A.; Wen, G.; Jiang, J.

    1999-01-01

    GLUT3 is expressed in rat muscle, but this glucose transporter protein has not been identified previously in adult human skeletal muscle. We quantified the rapidity of disappearance of mRNA and protein from human skeletal muscle at room temperature and at 4 degrees C. Fifty percent of the immunologically detectable GLUT3 protein disappeared by 1 hour at 20 degrees C and by 2 hours at 4 degrees C. mRNA for GLUT3 was decreased 50% by 2.2 hours at 20 degrees C and by 24 hours at 4 degrees C. Half of the measurable mRNAs for GLUT4, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), alpha-actin, and beta-myosin disappeared by 0.8 to 2.1 hours at 20 degrees C and by 5.0 to 16.6 hours at 4 degrees C. Previous conclusions that GLUT3 is not expressed in human muscle were likely drawn because of artifacts related to degradation of GLUT3 protein in the specimens prior to study. Because of the rapid degradation of protein and mRNA, autopsy specimens of muscle must be obtained within 6 hours of death, and even then, protein and mRNA data will likely dramatically underestimate their expression in fresh muscle. Some previously published conclusions and recommendations regarding autopsy specimens are not stringent enough to consistently yield useful protein and mRNA.

  14. Characterization of beta-defensin prepropeptide mRNA from chicken and turkey bone marrow.

    Science.gov (United States)

    Brockus, C W; Jackwood, M W; Harmon, B G

    1998-08-01

    Four avian beta-defension prepropeptide cDNA sequences [gallinacins: Gal 1 (synonym CHP 1, chicken heterophil peptide 1), and Gal 2; turkey heterophil peptides: THP 1 and THP 2] were amplified from chicken or turkey bone marrow mRNA samples, respectively. Partial chicken beta-defensin cDNA sequences were obtained using degenerate primers based on chicken peptide sequences (Gal 1/CHP 1 and Gal 2). The complete cDNA sequences of the chicken beta-defensins were then determined by designing specific intrapeptidal primers, from the newly acquired sequence, and pairing one primer with a specific poly A primer tail sequence (3' end) and the other primer with an adapter primer in a 5' rapid amplification of cDNA ends (RACE) reaction. The two, turkey beta-defensins were amplified from turkey marrow using primers designed from chicken beta-defensin preproregions. The complete amino acid sequences for the prepropeptides were deduced for all four avian beta-defensins. Previously, only partial mature peptide sequences for the turkey beta-defensins and complete mature peptide sequences for the chicken beta-defensins were known. All sequences obtained translated accurately to complete and partial amino acid sequences reported for beta-defensins purified from chicken and turkey heterophil granules except for one additional amino acid for Gal 1/CHP 1. The four deduced beta-defensin proregions lack the long, negatively charged propiece reported in classical defensin proregions. These regions are thought to stabilize and inactivate the positively charged mature peptide and target the propeptide to the storage granule. Instead, these beta-defensin proregions are shorter and similar to storage granule-free beta-defensins proregions reported for bovine tracheal antimicrobial peptide (TAP) and lingual antimicrobial peptide (LAP). These are the first prepropeptide beta-defensins from leukocyte granules to be completely characterized.

  15. Ranking Beta Sheet Topologies of Proteins

    DEFF Research Database (Denmark)

    Fonseca, Rasmus; Helles, Glennie; Winter, Pawel

    2010-01-01

    One of the challenges of protein structure prediction is to identify long-range interactions between amino acids.  To reliably predict such interactions, we enumerate, score and rank all beta-topologies (partitions of beta-strands into sheets, orderings of strands within sheets and orientations...... of paired strands) of a given protein.  We show that the beta-topology corresponding to the native structure is, with high probability, among the top-ranked. Since full enumeration is very time-consuming, we also suggest a method to deal with proteins with many beta-strands. The results reported...... in this paper are highly relevant for ab initio protein structure prediction methods based on decoy generation. The top-ranked beta-topologies can be used to find initial conformations from which conformational searches can be started. They can also be used to filter decoys by removing those with poorly...

  16. Analysis of correlations between protein complex and protein-protein interaction and mRNA expression

    Institute of Scientific and Technical Information of China (English)

    CAI Lun; XUE Hong; LU Hongchao; ZHAO Yi; ZHU Xiaopeng; BU Dongbo; LING Lunjiang; CHEN Runsheng

    2003-01-01

    Protein-protein interaction is a physical interaction of two proteins in living cells. In budding yeast Saccharomyces cerevisiae, large-scale protein-protein interaction data have been obtained through high-throughput yeast two-hybrid systems (Y2H) and protein complex purification techniques based on mass-spectrometry. Here, we collect 11855 interactions between total 2617 proteins. Through seriate genome-wide mRNA expression data, similarity between two genes could be measured. Protein complex data can also be obtained publicly and can be translated to pair relationship that any two proteins can only exist in the same complex or not. Analysis of protein complex data, protein-protein interaction data and mRNA expression data can elucidate correlations between them. The results show that proteins that have interactions or similar expression patterns have a higher possibility to be in the same protein complex than randomized selected proteins, and proteins which have interactions and similar expression patterns are even more possible to exist in the same protein complex. The work indicates that comprehensive integration and analysis of public large-scale bioinformatical data, such as protein complex data, protein-protein interaction data and mRNA expression data, may help to uncover their relationships and common biological information underlying these data. The strategies described here may help to integrate and analyze other functional genomic and proteomic data, such as gene expression profiling, protein-localization mapping and large-scale phenotypic data, both in yeast and in other organisms.

  17. Androgen receptor-beta mRNA levels in different tissues in breeding and post-breeding male and female sticklebacks, Gasterosteus aculeatus

    Directory of Open Access Journals (Sweden)

    Hoffmann Erik

    2012-03-01

    Full Text Available Abstract Background Androgens induce male characters by activating androgen receptors (AR. Previous quantitative studies on AR in fishes have been limited to few tissues and/or a single season/reproductive state. The aim of this investigation was to study the possible role of AR-beta expression levels in the control of male traits in the three-spined stickleback. To that end, AR-beta expression levels in major tissues in breeding and post-breeding male and female sticklebacks were examined. Methods AR-beta mRNA levels were quantified in ten tissues; eye, liver, axial muscle, heart, brain, intestine, ovary, testis, kidney and pectoral muscle in six breeding and post-breeding males and females using reverse transcription quantitative PCR. Results Breeding in contrast to post-breeding males built nests and showed secondary sexual characters (e.g. kidney hypertrophy and elevated androgen levels. Post-breeding females had lower ovarian weights and testosterone levels than breeding females. AR-beta was expressed in all studied tissues in both sexes and reproductive states with the highest expression in the gonads and in the kidneys. The kidney is an androgen target organ in sticklebacks, from which breeding males produce the protein spiggin, which is used in nest-building. There was also high AR-beta expression in the intestine, an organ that appears to take over hyperosmo-regulation in fresh water when the kidney hypertrophies in mature males and largely loses this function. The only tissue that showed effects of sex or reproductive state on AR-beta mRNA levels was the kidneys, where post-breeding males displayed higher AR-beta mRNA levels than breeding males. Conclusion The results indicate that changes in AR-beta mRNA levels play no or little role in changes in androgen dependent traits in the male stickleback.

  18. PLAC4 and beta-HCG mRNA levels are not altered in the maternal circulation of pregnancies with trisomy 21.

    Science.gov (United States)

    Banzola, Irina; Rusterholz, Corinne; Zannoni, Letizia; Rizzo, Nicola; Zhong, Xiao Yan; Caramelli, Elisabetta; Holzgreve, Wolfgang; Farina, Antonio; Hahn, Sinuhe

    2008-12-01

    Beta-human chorionic gonadotropin (HCG) and pregnancy-associated plasma protein (PAPP-A) are placentally produced proteins whose levels are altered in pregnancies with trisomy 21. PLAC4 is located on chromosome 21 and its expression is restricted to the placenta. Here we investigated whether the levels of beta-HCG-, PAPP-A- and PLAC4 mRNA could be able to discriminate pregnancies whose fetus is affected by trisomy 21. Hundred and forty-three blood samples from normal pregnancies and eight samples from trisomic pregnancies were collected. Total RNA was extracted from whole maternal blood, reverse-transcribed and the three mRNAs were quantified by real-time quantitative PCR. Hundred and nine controls were also tested for the serum levels of PAPP-A and HCG proteins. Beta-HCG and PLAC4 mRNAs were detected in all samples, in higher amounts than in plasma, whereas the detection rate for PAPP-A mRNA was below 10%. The levels of beta-HCG mRNA significantly correlated with the circulatory concentrations of the HCG protein. However, neither beta-HCG- nor PLAC4 mRNAs show a significant difference between cases and controls. Maternal blood levels of beta-HCG-, PLAC4- and PAPP-A mRNAs are not useful markers for the screening of pregnancies with trisomy 21 as their concentrations are either not significantly altered (beta-HCG and PLAC4) or too low to be detected (PAPP-A).

  19. Aβ对小胶质细胞中IL-1β及iNOS mRNA水平的影响%The changes of IL-1β and iNOS mRNA in cultured hippocampal microglia of rats exposed to amyloid beta-protein

    Institute of Scientific and Technical Information of China (English)

    贾丽艳; 拓西平; 朱嘉琦; 周俊

    2005-01-01

    目的观察β-淀粉样蛋白(amyloid beta-protein, Aβ)对原代培养的大鼠海马小胶质细胞中白细胞介素-1β(interleukin-1 beta ,IL-1β)及诱导型一氧化氮合酶(inducible nitric oxide synthase, iNOS)mRNA 水平的影响,探讨Aβ诱导的氧化应激和炎症反应在阿尔茨海默病(Alzheimer's disease, AD)发病机制中的作用. 方法采用Aβ25-35孵育原代培养的新生大鼠海马小胶质细胞的方法建立Aβ诱导损伤的神经细胞模型,用半定量逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction, RT-PCR)方法观察体外条件下Aβ25-35对小胶质细胞中IL-1β及iNOS mRNA 水平的影响. 结果 1 μmol/L Aβ25-35作用于原代培养的新生大鼠海马小胶质细胞 48 h后,IL-1β及iNOS mRNA 水平较空白对照组显著升高(P<0.01). 结论 Aβ25-35诱导的氧化应激和炎症反应在AD的发病机制中具有重要作用.

  20. Role of CC chemokines (macrophage inflammatory protein-1 beta, monocyte chemoattractant protein-1, RANTES) in acute lung injury in rats

    DEFF Research Database (Denmark)

    Bless, N M; Huber-Lang, M; Guo, R F

    2000-01-01

    were cloned, the proteins were expressed, and neutralizing Abs were developed. mRNA and protein expression for MIP-1 beta and MCP-1 were up-regulated during the inflammatory response, while mRNA and protein expression for RANTES were constitutive and unchanged during the inflammatory response...... that in chemokine-dependent inflammatory responses in lung CC chemokines do not necessarily demonstrate redundant function.......The role of the CC chemokines, macrophage inflammatory protein-1 beta (MIP-1 beta), monocyte chemotactic peptide-1 (MCP-1), and RANTES, in acute lung inflammatory injury induced by intrapulmonary deposition of IgG immune complexes injury in rats was determined. Rat MIP-1 beta, MCP-1, and RANTES...

  1. Rev-erb beta regulates the Srebp-1c promoter and mRNA expression in skeletal muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Ramakrishnan, Sathiya N.; Lau, Patrick; Crowther, Lisa M. [The University of Queensland, Institute for Molecular Bioscience, St. Lucia, Qld 4072 (Australia); Cleasby, Mark E. [Diabetes and Obesity Research Program, Garvan Institute of Medical Research, St. Vincent' s Hospital, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010 (Australia); Millard, Susan; Leong, Gary M. [The University of Queensland, Institute for Molecular Bioscience, St. Lucia, Qld 4072 (Australia); Cooney, Gregory J. [Diabetes and Obesity Research Program, Garvan Institute of Medical Research, St. Vincent' s Hospital, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010 (Australia); Muscat, George E.O., E-mail: g.muscat@imb.uq.edu.au [The University of Queensland, Institute for Molecular Bioscience, St. Lucia, Qld 4072 (Australia)

    2009-10-30

    The nuclear hormone receptor, Rev-erb beta operates as a transcriptional silencer. We previously demonstrated that exogenous expression of Rev-erb{beta}{Delta}E in skeletal muscle cells increased Srebp-1c mRNA expression. We validated these in vitro observations by injection of an expression vector driving Rev-erb{beta}{Delta}E expression into mouse tibialis muscle that resulted in increased Srebp-1c mRNA expression. Paradoxically, Rev-erb{beta} siRNA expression in skeletal muscle cells repressed Srebp-1c expression, and indicated that Rev-erb{beta} expression was necessary for Srebp-1c expression. ChIP analysis demonstrated that Rev-erb{beta} was recruited to the Srebp-1c promoter. Moreover, Rev-erb{beta} trans-activated the Srebp-1c promoter, in contrast, Rev-erb{beta} efficiently repressed the Rev-erb{alpha} promoter, a previously characterized target gene. Finally, treatment with the Rev-erb agonist (hemin) (i) increased the trans-activation of the Srebp-1c promoter by Rev-erb{beta}; and (ii) increased Rev-erb{beta} and Srebp-1c mRNA expression. These data suggest that Rev-erb{beta} has the potential to activate gene expression, and is a positive regulator of Srebp-1c, a regulator of lipogenesis.

  2. Protein Structure and the Sequential Structure of mRNA

    DEFF Research Database (Denmark)

    Brunak, Søren; Engelbrecht, Jacob

    1996-01-01

    A direct comparison of experimentally determined protein structures and their corresponding protein coding mRNA sequences has been performed, We examine whether real world data support the hypothesis that clusters of rare codons correlate with the location of structural units in the resulting pro...... nucleotide features of 16S-like ribosomal RNA sequences and related to mechanisms for maintaining the correct reading frame by the ribosome. (C) 1996 Wiley-Liss, Inc....

  3. Effect of acidic ribosomal phosphoprotein mRNA 5'-untranslated region on gene expression and protein accumulation.

    Science.gov (United States)

    Bermejo, B; Remacha, M; Ortiz-Reyes, B; Santos, C; Ballesta, J P

    1994-02-11

    Constructions were made from genes encoding ribosomal acidic phosphoproteins YP1 beta (L44') and YP2 beta (L45) from Saccharomyces cerevisiae in which different parts of the 5'-untranslated regions were included. The constructs were inserted into centromeric plasmids under the control of the GAL1 promoter and expressed in yeast strains in which the genes coding for each acidic protein family, P1 and P2, had been disrupted. Deletions in the 5' region of the two genes have been found to oppositely affect their expression. Deletion of most of this region strongly stimulates the expression of YP2 beta (L45), increasing the translation efficiency of the mRNA, and generating a 6-fold excess of protein in the cell. A similar deletion in the rpYP1 beta gene represses the expression of the protein, reducing drastically the amount of the mRNA in the cell. The overexpression of rpYP2 beta affects the cell growth by inhibiting protein synthesis at the level of initiation. Reduction of the YP2 beta(L45) overproduction by growing in controlled concentrations of glucose abolishes the inhibitory effect. The excess protein, probably as a high molecular weight complex, apparently interferes with the joining of the 60 S subunit to the initiation complex generating the accumulation of polysome half-mers. In addition, the results indicate the existence of a regulatory mechanism by which each one of the two acidic proteins controls the expression of the other polypeptide. YP1 beta(L44') represses the expression of YP2 beta(L45), while this protein stimulates the expression of YP1 beta(L44').

  4. Expression of Endogenous Beta Retroviruses and Hyal-2 mRNA in Immune Organs of Fetuses and Lambs

    Institute of Scientific and Technical Information of China (English)

    Jing-wei Qi; Xiao-li Wu; Shu-ying Liu; Gui-fang Cao

    2012-01-01

    Endogenous beta retroviruses (enJSRV) are highly homologous with Jaagsiekte sheep retrovirus (exJSRV),this exogenous retrovirus is the aetiological agent of ovine pulmonary adenocarcinoma (OPA).The aim of this study was to clarify the function of enJSRV and the immunological mechanisms of its corresponding antibody,that is undetectable in JSRV-infected ovine serum.The expression of enJSRV envelope protein and Hyal-2 mRNA in immune organs and lungs of ovine fetuses and lambs were analyzed by Real-Time reverse transcription PCR and In Situ Hybridization using specific probes.In Situ Hybridization results indicated that the enJSRV envelope protein and Hyal-2 mRNA were expressed in thymus,spleen,mesenteric lymph nodes and lungs at different times,while no positive signals were detected in the negative controls.On the other hand,results from Real-Time reverse transcription PCR analysis showed that in 130d fetuses and 3d newborn lambs the enJSRV mRNA levels were much higher in organs associated with the immune system than that in lungs,especially in the thymus and spleen,but levels of Hyal-2 mRNA expression was not significantly different in all collected tissue.These results provided evidence from an immunology point of view to understand why the circulating antibodies against exJSRV are undetectable in JSRV-infected ovine,and will help to unravel the pathogenesis of JSRV-infected ovine.

  5. Phospholipase C-{delta}{sub 1} regulates interleukin-1{beta} and tumor necrosis factor-{alpha} mRNA expression

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Eric; Jakinovich, Paul; Bae, Aekyung [Department of Anesthesiology, Health Sciences Center L4 Rm 081, Stony Brook University, Stony Brook, NY 11794 (United States); Rebecchi, Mario, E-mail: Mario.rebecchi@SBUmed.org [Department of Anesthesiology, Health Sciences Center L4 Rm 081, Stony Brook University, Stony Brook, NY 11794 (United States)

    2012-10-01

    Phospholipase C-{delta}{sub 1} (PLC{delta}{sub 1}) is a widely expressed highly active PLC isoform, modulated by Ca{sup 2+} that appears to operate downstream from receptor signaling and has been linked to regulation of cytokine production. Here we investigated whether PLC{delta}{sub 1} modulated expression of the pro-inflammatory cytokines interleukin-1{beta} (IL-1{beta}), tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-6 (IL-6) in rat C6 glioma cells. Expression of PLC{delta}{sub 1} was specifically suppressed by small interfering RNA (siRNA) and the effects on cytokine mRNA expression, stimulated by the Toll-like receptor (TLR) agonist, lipopolysaccharide (LPS), were examined. Real-time polymerase chain reaction (RT-PCR) results showed that PLC{delta}{sub 1} knockdown enhanced expression IL-1{beta} and tumor necrosis factor-{alpha} (TNF-{alpha}) mRNA by at least 100 fold after 4 h of LPS stimulation compared to control siRNA treatment. PLC{delta}{sub 1} knock down caused persistently high Nf{kappa}b levels at 4 h of LPS stimulation compared to control siRNA-treated cells. PLC{delta}{sub 1} knockdown was also associated with elevated nuclear levels of c-Jun after 30 min of LPS stimulation, but did not affect LPS-stimulated p38 or p42/44 MAPK phosphorylation, normally associated with TLR activation of cytokine gene expression; rather, enhanced protein kinase C (PKC) phosphorylation of cellular proteins was observed in the absence of LPS stimulation. An inhibitor of PKC, bisindolylmaleimide II (BIM), reversed phosphorylation, prevented elevation of nuclear c-Jun levels, and inhibited LPS-induced increases of IL-1{beta} and TNF-{alpha} mRNA's induced by PLC{delta}{sub 1} knockdown. Our results show that loss of PLC{delta}{sub 1} enhances PKC/c-Jun signaling and up-modulates pro-inflammatory cytokine gene transcription in concert with the TLR-stimulated p38MAPK/Nf{kappa}b pathway. Our findings are consistent with the idea that PLC{delta}{sub 1} is a

  6. Inhibition of beta cell growth and function by bone morphogenetic proteins

    DEFF Research Database (Denmark)

    Bruun, Christine; Christensen, Gitte Lund; Jacobsen, Marie L B

    2014-01-01

    of diabetes, there is an increase in the expression of inhibitory factors that prevent the beta cells from adapting to the increased need for insulin. We evaluated the effects of bone morphogenetic protein (BMP) 2 and -4 on beta cells. METHODS: The effects of BMP2 and -4 on beta cell proliferation, apoptosis......: BMP2 and -4 were found to inhibit basal as well as growth factor-stimulated proliferation of primary beta cells from rats and mice. Bmp2 and Bmp4 mRNA and protein were expressed in islets and regulated by inflammatory cytokines. Neutralisation of endogenous BMP activity resulted in enhanced....../INTERPRETATION: These data show that BMP2 and -4 exert inhibitory actions on beta cells in vitro and suggest that BMPs exert regulatory roles of beta cell growth and function....

  7. Imaging mRNA and protein interactions within neurons

    Science.gov (United States)

    Eliscovich, Carolina; Shenoy, Shailesh M.

    2017-01-01

    RNA–protein interactions are essential for proper gene expression regulation, particularly in neurons with unique spatial constraints. Currently, these interactions are defined biochemically, but a method is needed to evaluate them quantitatively within morphological context. Colocalization of two-color labels using wide-field microscopy is a method to infer these interactions. However, because of chromatic aberrations in the objective lens, this approach lacks the resolution to determine whether two molecules are physically in contact or simply nearby by chance. Here, we developed a robust super registration methodology that corrected the chromatic aberration across the entire image field to within 10 nm, which is capable of determining whether two molecules are physically interacting or simply in proximity by random chance. We applied this approach to image single-molecule FISH in combination with immunofluorescence (smFISH-IF) and determined whether the association between an mRNA and binding protein(s) within a neuron was significant or accidental. We evaluated several mRNA-binding proteins identified from RNA pulldown assays to determine which of these exhibit bona fide interactions. Surprisingly, many known mRNA-binding proteins did not bind the mRNA in situ, indicating that adventitious interactions are significant using existing technology. This method provides an ability to evaluate two-color registration compatible with the scale of molecular interactions. PMID:28223507

  8. 14-3-3 protein binds to the low molecular weight neurofilament (NFL) mRNA 3' UTR.

    Science.gov (United States)

    Ge, Wei-Wen; Volkening, Kathryn; Leystra-Lantz, Cheryl; Jaffe, Howard; Strong, Michael J

    2007-01-01

    We have previously reported that altered stability of low molecular weight neurofilament (NFL) mRNA in lumbar spinal cord homogenates in amyotrophic lateral sclerosis (ALS) is associated with altered expression of trans-acting 3' UTR mRNA binding proteins. We have identified two hexanucleotide motifs as the main cis elements and, using LC/MS/MS of peptide digests of NFL 3' UTR interacting proteins from human spinal cord, observed that 14-3-3 proteins interact with these motifs. 14-3-3 beta, zeta, tau, gamma, and eta isoforms were found to be expressed in human spinal cord. Each isoform was expressed in vitro and shown to interact with NFL 3' UTR mRNA. Mutation of one or both motifs resulted in decreased 14-3-3 interaction, changes in predicted mRNA structure or alteration in stability of the mRNA. These data show a novel interaction for 14-3-3 with NFL mRNA, and suggests that 14-3-3 may play a role in regulating NFL mRNA stability.

  9. AU-rich elements and alternative splicing in the beta-catenin 3'UTR can influence the human beta-catenin mRNA stability.

    NARCIS (Netherlands)

    Thiele, A.; Nagamine, Y.; Hauschildt, S.; Clevers, J.C.

    2006-01-01

    Beta-catenin, the central player of the Wnt signaling cascade, is a well-known oncogene. The regulation of beta-catenin protein stability has been studied extensively while other mechanisms that control cellular levels of beta-catenin have hardly been addressed. In this study, we show that there are

  10. Reductions in laminin beta2 mRNA translation are responsible for impaired IGFBP-5-mediated mesangial cell migration in the presence of high glucose.

    Science.gov (United States)

    Schaeffer, Valerie; Hansen, Kim M; Morris, David R; Abrass, Christine K

    2010-02-01

    Insulin-like growth factor binding protein-5 (IGFBP-5) mediates mesangial cell migration through activation of cdc42, and laminin421 binding to alpha(6)beta(1)-integrin (Berfield AK, Hansen KM, Abrass CK. Am J Physiol Cell Physiol 291: C589-C599, 2006). Because glomerular expression of laminin beta(2) is reduced in diabetic rats (Abrass CK, Spicer D, Berfield AK, St. John PL, Abrahamson DR. Am J Pathol 151: 1131-1140, 1997), we directly examined the effect of hyperglycemia on mesangial cell migration and laminin beta2 expression. Migration mediated by IGFBP-5 is impaired in the presence of 25 mM glucose. This reduction in migration was found to result from a loss in mesangial cell synthesis of laminin421, and IGFBP-5-induced migration could be restored by replacing laminin421. Additional studies showed that there was selective reduction in mRNA translation of laminin beta2 in the presence of high glucose. Preserved synthesis of laminin beta1 indicates that not all proteins are reduced by high glucose and confirms prior data showing that laminin411 cannot substitute for laminin421 in IGFBP-5-mediated migration. Given the importance of mesangial migration in the reparative response to diabetes-associated mesangiolysis, these findings provide new insights into abnormalities associated with diabetic nephropathy and the potential importance of differential control of protein translation in determination of alterations of protein expression.

  11. Platelets possess functional TGF-beta receptors and Smad2 protein.

    Science.gov (United States)

    Lev, P R; Salim, J P; Marta, R F; Osorio, M J Mela; Goette, N P; Molinas, F C

    2007-02-01

    TGF-beta1 plays a main role in tissue repair by regulating extracellular matrix production and tissue granulation. Platelets are one of the main sources of this cytokine in the circulation. The aim of this study was to evaluate the presence of the TGF-beta receptors on platelets, the effect of TGF-beta1 on platelet aggregation and the underlying intracellular mechanisms. TGF-beta receptors on platelets were studied by flow cytometry and their mRNA by PCR. Platelet aggregation was assessed by turbidimetric methods and intracellular pathways by Western blot. TGF-beta receptor type II and mRNA codifying for TbetaRI and TbetaRII were found in platelets. We demonstrated that TGF-beta1 did not trigger platelet aggregation by itself but had a modulating effect on ADP-induced platelet aggregation. Either inhibition or increase in platelet aggregation, depending on the exposure time to TGF-beta1 and the ADP concentration used, were shown. We found that platelets possess Smad2 protein and that its phosphorylation state is increased after exposure to TGF-beta1. Besides, TGF-beta1 modified the pattern of ADP-induced tyrosine phosphorylation. Increased phosphorylation levels of 64-, 80- and 125-kDa proteins during short time incubation with TGF-beta1 and increased phosphorylation of 64- and 125-kDa proteins after longer incubation were observed. The modulating effect of TGF-beta1 on platelet aggregation could play a role during pathological states in which circulating TGF-beta1 levels are increased and intravascular platelet activation is present, such as myeloproliferative disorders. In vascular injury, in which platelet activation followed by granule release generates high local ADP concentrations, it could function as a physiological mechanism of platelet activation control.

  12. Cytokines in relapsing experimental autoimmune encephalomyelitis in DA rats: persistent mRNA expression of proinflammatory cytokines and absent expression of interleukin-10 and transforming growth factor-beta

    DEFF Research Database (Denmark)

    Issazadeh-Navikas, Shohreh; Lorentzen, J C; Mustafa, M I;

    1996-01-01

    ) of diseased DA rats. We demonstrate that peripheral lymphoid cells stimulated in vitro with encephalitogenic peptides 69-87 and 87-101 of myelin basic protein responded with high mRNA expression for proinflammatory cytokines; interferon-gamma, interleukin-12 (IL-12), tumour necrosis factors alpha and beta, IL......-1 beta and cytolysin. A high expression of mRNA for these proinflammatory cytokines was also observed in the CNS where it was accompanied by classical signs of inflammation such as expression of major histocompatibility complex class I and II, CD4, CD8 and IL-2 receptor. The expression of m......RNA for proinflammatory cytokines was remarkably long-lasting in DA rats as compared to LEW rats which display a brief and monophasic EAE. Furthermore, mRNAs for putative immunodownmodulatory cytokines, i.e. transforming growth factor-beta (TGF-beta), IL-10 and IL-4 were almost absent in DA rats, in both the CNS...

  13. Isolation and characterization of BetaM protein encoded by ATP1B4 - a unique member of the Na,K-ATPase {beta}-subunit gene family

    Energy Technology Data Exchange (ETDEWEB)

    Pestov, Nikolay B. [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States); Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997 (Russian Federation); Zhao, Hao [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States); Basrur, Venkatesha [Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109 (United States); Modyanov, Nikolai N., E-mail: nikolai.modyanov@utoledo.edu [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States)

    2011-09-09

    Highlights: {yields} Structural properties of BetaM and Na,K-ATPase {beta}-subunits are sharply different. {yields} BetaM protein is concentrated in nuclear membrane of skeletal myocytes. {yields} BetaM does not associate with a Na,K-ATPase {alpha}-subunit in skeletal muscle. {yields} Polypeptide chain of the native BetaM is highly sensitive to endogenous proteases. {yields} BetaM in neonatal muscle is a product of alternative splice mRNA variant B. -- Abstract: ATP1B4 genes represent a rare instance of the orthologous gene co-option that radically changed functions of encoded BetaM proteins during vertebrate evolution. In lower vertebrates, this protein is a {beta}-subunit of Na,K-ATPase located in the cell membrane. In placental mammals, BetaM completely lost its ancestral role and through acquisition of two extended Glu-rich clusters into the N-terminal domain gained entirely new properties as a muscle-specific protein of the inner nuclear membrane possessing the ability to regulate gene expression. Strict temporal regulation of BetaM expression, which is the highest in late fetal and early postnatal myocytes, indicates that it plays an essential role in perinatal development. Here we report the first structural characterization of the native eutherian BetaM protein. It should be noted that, in contrast to structurally related Na,K-ATPase {beta}-subunits, the polypeptide chain of BetaM is highly sensitive to endogenous proteases that greatly complicated its isolation. Nevertheless, using a complex of protease inhibitors, a sample of authentic BetaM was isolated from pig neonatal skeletal muscle by a combination of ion-exchange and lectin-affinity chromatography followed by SDS-PAGE. Results of the analysis of the BetaM tryptic digest using MALDI-TOF and ESI-MS/MS mass spectrometry have demonstrated that native BetaM in neonatal skeletal muscle is a product of alternative splice mRNA variant B and comprised of 351 amino acid residues. Isolated BetaM protein was

  14. Differentiation-dependent expression of retinoid-binding proteins in BFC-1 beta adipocytes.

    Science.gov (United States)

    Zovich, D C; Orologa, A; Okuno, M; Kong, L W; Talmage, D A; Piantedosi, R; Goodman, D S; Blaner, W S

    1992-07-15

    Recently, we demonstrated that adipose tissue plays an important role in retinol storage and retinol-binding protein (RBP) synthesis. Our data suggested that RBP expression in adipose tissue is dependent on the state of adipocyte differentiation. To examine this possibility, we explored the differentiation-dependent expression of RBP using BFC-1 beta preadipocytes, which can be stimulated to undergo adipose differentiation. Total RNA was isolated from undifferentiated (preadipocytes) and differentiated (adipocytes) BFC-1 beta cells and analyzed by Northern blotting. RBP mRNA was not detected in the preadipocytes, but considerable RBP mRNA was present in differentiated BFC-1 beta cells. In BFC-1 beta cells, induced to differentiate with insulin and thyroid hormone, RBP mRNA was first detected after 4 days, reached a maximum level by day 10, and remained at this maximum level for at least 2 more days. Cellular retinol-binding protein was expressed at low levels in the BFC-1 beta preadipocytes and the level of expression increased for 6 days after induction to differentiate and slowly declined on later days. Neither the maximum level of RBP expression nor the day on which this level was reached was influenced by the level of retinol provided in the BFC-1 beta culture medium. BFC-1 beta cells secreted newly synthesized RBP into the culture medium at a rate of 43 +/- 14 ng RBP/24 h/10(6) adipocytes. When the BFC-1 beta adipocytes were provided 1.0 microM retinol in the medium, they accumulated the retinol and synthesized retinyl esters. These studies with BFC-1 beta cells confirm that RBP synthesis and secretion and retinol accumulation are intrinsic properties of differentiated adipocytes. Furthermore, they suggest that RBP and cellular retinol-binding protein gene expression are regulated as part of a package of genes which are modulated during adipocyte differentiation.

  15. Induction of aromatic L-amino acid decarboxylase mRNA by interleukin-1 beta and prostaglandin E2 in PC12 cells.

    Science.gov (United States)

    Li, X M; Juorio, A V; Boulton, A A

    1994-05-01

    Aromatic 1-amino acid decarboxylase (AADC) is involved in the synthesis of the putative neurotransmitters dopamine (DA), norepinephrine (NA) and 5-hydroxytryptamine (5-HT). We report here that the gene expression of AADC can be regulated by interleukin (IL) 1-beta and prostaglandin (PG) E2 in PC12 cells. The cells were treated with different doses of IL 1-beta and PGE2 for 3 days. Slot blot hybridization was performed to detect AADC mRNA and Western immunoblot to detect AADC protein. The cDNA probe for rat AADC was generated by the PCR method. IL 1-beta and PGE2 produced a dose- and time-dependent up-regulation in AADC mRNA levels (up to 200% of the control values) which was followed by a stable increase in AADC protein. The data further support the suggestion that AADC is a regulated enzyme and that the regulation occurs at the level of gene expression. Because IL-1 is synthesized, and acts locally, within the brain to influence neuronal and glial functions, it has been proposed to be a mediator with both beneficial and detrimental responses to inflammation and injury. The regulation of AADC by IL-1 may indicate a possible involvement for AADC in neuronal injury and recovery. Since IL-1 promotes PGE2 formation, its effects may be occurring by increasing level of PGE2.

  16. Quantitative analysis of transforming growth factor beta 1 mRNA in patients with alcoholic liver disease

    Institute of Scientific and Technical Information of China (English)

    Wei-Xing Chen; You-Ming Li; Chao-Hui Yu; Wei-Min Cai; Min Zheng; Feng Chen

    2002-01-01

    AIM: To investigate the expression of the transforminggrowth factor beta 1 (TGF- beta 1 ) mRNA in different stagesof alcoholic liver disease (ALD) and its clinical value.METHODS: One hundred and seven male alcoholics weregrouped by clinical findings into four groups: alcoholabusers without liver impairment (n=22 ), alcoholicsteatosis ( n = 30 ); alcoholic hepatitis ( n = 31 ); andalcoholic cirrhosis ( n = 24 ) Using peripheral bloodmononuclear cells(PBMC) as samples the gene expressionof TGF-beta 1 was examined quantitatively by reversetranscription polymerase chain reaction (RT-PCR) and dotblot. There are 34 healthy subjects served as control.RESULTS: The expression of TGF-beta 1 from all ALDpatients was significantly greater than that in controls ( 1. 320± 1.162 vs 0.808±0.276, P<0.001). The differences of theexpressions were significant between the patients from eachgroups ( alcoholic steatosis, alcoholic hepatitis andalcoholic cirrhosis) and the controls ( 1. 168 ± 0.852, 1.462 ±1.657, 1.329± 0.610 vs 0.808 ± 0.276, P< 0.050). Nosignificant differences of TGF -beta 1 mRNA expression wereobserved between alcohol abusers without liver impairmentand controls. The expressions in patients with alcoholichepatitis and alcoholic cirrhosis were significantly greaterthan that in alcohol abusers respectively (1.462 ± 1. 657, 1.329 ± 0. 610 vs 0. 841 ± 0. 706, P < 0. 050). No significantdifferences of TGF -beta 1 mRNA expression were observedbetween alcoholic fatty liver men and alcohol abusers.CONCLUSION: TGF-beta 1 expression level can be a riskfactor for alcoholic liver disease and might be related to theinflammatory activity and fibrosis of the liver in patients .

  17. Role of highly conserved pyrimidine-rich sequences in the 3' untranslated region of the GAP-43 mRNA in mRNA stability and RNA-protein interactions.

    Science.gov (United States)

    Kohn, D T; Tsai, K C; Cansino, V V; Neve, R L; Perrone-Bizzozero, N I

    1996-03-01

    We have shown previously that the mRNA for the growth-associated protein GAP-43 is selectively stabilized during neuronal differentiation. In this study, we explored the role of its highly conserved 3' untranslated region (3'UTR) in mRNA stability and RNA-protein interactions. The 3'UTRs of the rat and chicken GAP-43 mRNAs show 78% sequence identity, which is equivalent to the conservation of their coding regions. In rat PC12 cells stably transfected with the full-length rat or chicken GAP-43 cDNAs, the transgene mRNAs decayed with same half-life of about 3 h. The GAP-43 3'UTR also caused the rabbit beta-globin mRNA to decay with a half-life of 4 h, indicating that the major determinants for GAP-43 mRNA stability are localized in its highly conserved 3'UTR. Three brain cytosolic RNA-binding proteins (molecular mass 40, 65 and 95 kDa) were found to interact with both the rat and chicken GAP-43 mRNAs. These RNA-protein interactions were specific and involved pyrimidine-rich sequences in the 3'UTR. Like the GAP-43 mRNA, the activity of these proteins was enriched in brain and increased during development. We propose that highly conserved pyrimidine-rich sequences in the 3'UTR of this mRNA regulate GAP-43 gene expression via interactions with specific RNA-binding proteins.

  18. Correlation of mRNA and protein in complex biological samples.

    Science.gov (United States)

    Maier, Tobias; Güell, Marc; Serrano, Luis

    2009-12-17

    The correlation between mRNA and protein abundances in the cell has been reported to be notoriously poor. Recent technological advances in the quantitative analysis of mRNA and protein species in complex samples allow the detailed analysis of this pathway at the center of biological systems. We give an overview of available methods for the identification and quantification of free and ribosome-bound mRNA, protein abundances and individual protein turnover rates. We review available literature on the correlation of mRNA and protein abundances and discuss biological and technical parameters influencing the correlation of these central biological molecules.

  19. Fragile X mental retardation protein control of neuronal mRNA metabolism: Insights into mRNA stability.

    Science.gov (United States)

    De Rubeis, Silvia; Bagni, Claudia

    2010-01-01

    The fragile X mental retardation protein (FMRP) is an RNA binding protein that has an essential role in neurons. From the soma to the synapse, FMRP is associated with a specific subset of messenger RNAs and controls their posttranscriptional fates, i.e., dendritic localization and local translation. Because FMRP target mRNAs encode important neuronal proteins, the deregulation of their expression in the absence of FMRP leads to a strong impairment of synaptic function. Here, we review emerging evidence indicating a critical role for FMRP in the control of mRNA stability. To date, two mRNAs have been identified as being regulated in this manner: PSD-95 mRNA, encoding a scaffolding protein, and Nxf1 mRNA, encoding a general export factor. Moreover, expression studies suggest that the turnover of other neuronal mRNAs, including those encoding for the GABA(A) receptors subunits, could be affected by the loss of FMRP. According to the specific target and/or cellular context, FMRP could influence mRNA stability in the brain. Copyright 2009 Elsevier Inc. All rights reserved.

  20. Squid Giant Axon Contains Neurofilament Protein mRNA but does not Synthesize Neurofilament Proteins.

    Science.gov (United States)

    Gainer, Harold; House, Shirley; Kim, Dong Sun; Chin, Hemin; Pant, Harish C

    2017-04-01

    When isolated squid giant axons are incubated in radioactive amino acids, abundant newly synthesized proteins are found in the axoplasm. These proteins are translated in the adaxonal Schwann cells and subsequently transferred into the giant axon. The question as to whether any de novo protein synthesis occurs in the giant axon itself is difficult to resolve because the small contribution of the proteins possibly synthesized intra-axonally is not easily distinguished from the large amounts of the proteins being supplied from the Schwann cells. In this paper, we reexamine this issue by studying the synthesis of endogenous neurofilament (NF) proteins in the axon. Our laboratory previously showed that NF mRNA and protein are present in the squid giant axon, but not in the surrounding adaxonal glia. Therefore, if the isolated squid axon could be shown to contain newly synthesized NF protein de novo, it could not arise from the adaxonal glia. The results of experiments in this paper show that abundant 3H-labeled NF protein is synthesized in the squid giant fiber lobe containing the giant axon's neuronal cell bodies, but despite the presence of NF mRNA in the giant axon no labeled NF protein is detected in the giant axon. This lends support to the glia-axon protein transfer hypothesis which posits that the squid giant axon obtains newly synthesized protein by Schwann cell transfer and not through intra-axonal protein synthesis, and further suggests that the NF mRNA in the axon is in a translationally repressed state.

  1. mRNA and Protein levels of rat pancreas specific protein disulphide isomerase are downregulated during Hyperglycemia.

    Science.gov (United States)

    Gupta, Rajani; Bhar, Kaushik; Sen, Nandini; Bhowmick, Debajit; Mukhopadhyay, Satinath; Panda, Koustubh; Siddhanta, Anirban

    2016-02-01

    Diabetes (Type I and Type II) which affects nearly every organ in the body is a multi-factorial non-communicable disorder. Hyperglycemia is the most characteristic feature of this disease. Loss of beta cells is common in both types of diabetes whose detailed cellular and molecular mechanisms are yet to be elucidated. As this disease is complex, identification of specific biomarkers for its early detection, management and devising new therapies is challenging. Based on the fact that functionally defective proteins provide the biochemical basis for many diseases, in this study, we tried to identify differentially expressed proteins during hyperglycemia. For that, hyperglycemia was induced in overnight fasted rats by intra-peritoneal injection of streptozotocin (STZ). The pancreas was isolated from control and treated rats for subsequent analyses. The 2D-gel electrophoresis followed by MALDI-TOF-MS-MS analyses revealed several up- and down-regulated proteins in hyperglycemic rat pancreas including the downregulation of a pancreas specific isoform of protein disulphide isomerase a2 (Pdia2).This observation was validated by western blot. Quantitative PCR experiments showed that the level of Pdia2 mRNA is also proportionally reduced in hyperglycemic pancreas.

  2. Prostaglandin E2 stimulates S100A8 expression by activating protein kinase A and CCAAT/enhancer-binding-protein-beta in prostate cancer cells.

    Science.gov (United States)

    Miao, Lin; Grebhardt, Sina; Shi, Jiandang; Peipe, Isabelle; Zhang, Ju; Mayer, Doris

    2012-11-01

    S100A8 and S100A9 are strongly expressed in epithelial cells of human prostate cancer. However, the regulation of their expression is unclear. Here we show that S100A8 and to a lesser extent S100A9 mRNA expression is induced by prostaglandin E2 in a dose and time-dependent manner in PC-3 prostate cancer cells as well as in BPH-1 benign prostatic epithelial cells. Prostanoid receptor EP2 antagonist AH6809 and EP4 antagonist AH23848, as well as protein kinase A inhibitor H89, inhibited prostaglandin E2 mediated increase in S100A8 mRNA expression as well as promoter activity. Sequence analysis detected a potential binding site of the transcription factor CCAAT/enhancer-binding-protein-beta within the proximal S100A8 promoter. CCAAT/enhancer-binding-protein-beta overexpression increased S100A8 mRNA and protein expression as well as its promoter activity. The latter was prevented by mutation of the potential CCAAT/enhancer-binding-protein-beta binding site within the S100A8 promoter. Chromatin immunoprecipitation revealed increased binding of CCAAT/enhancer-binding-protein-beta to the S100A8 promoter in prostaglandin E2 treated cells. Knockdown of CCAAT/enhancer-binding-protein-beta by siRNA blocked prostaglandin E2 mediated induction of S100A8 promoter activity and mRNA expression. Our results indicate that in prostate cancer cells, S100A8 expression is stimulated by prostaglandin E2 via EP2 and EP4 receptors through activation of the protein kinase A signaling pathway and subsequent stimulation of CCAAT/enhancer-binding-protein-beta binding to the S100A8 promoter.

  3. Alternative mRNA Splicing from the Glial Fibrillary Acidic Protein (GFAP) Gene Generates Isoforms with Distinct Subcellular mRNA Localization Patterns in Astrocytes

    DEFF Research Database (Denmark)

    Thomsen, Rune; Daugaard, Tina Fuglsang; Holm, Ida E;

    2013-01-01

    The intermediate filament network of astrocytes includes Glial fibrillary acidic protein (Gfap) as a major component. Gfap mRNA is alternatively spliced resulting in generation of different protein isoforms where Gfapa is the most predominant isoform. The Gfapd isoform is expressed in proliferating......RNA localization patterns were dependent on the different 39-exon sequences included in Gfapd and Gfapa mRNA. The presented results show that alternative Gfap mRNA splicing results in isoform-specific mRNA localization patterns with resulting different local mRNA concentration ratios which have potential...

  4. Bacterial infection of osteoblasts induces interleukin-1beta and interleukin-18 transcription but not protein synthesis.

    Science.gov (United States)

    Marriott, Ian; Hughes, Francis M; Bost, Kenneth L

    2002-10-01

    A growing body of evidence has shown that bacterially challenged bone-forming osteoblasts are a significant source of an array of cytokines and chemokines that can support immune responses during bone disease. In the present study, Staphylococcus aureus and Salmonella, two common pathogens of bone, were investigated for their ability to induce production of two related inflammatory cytokines, interleukin-1beta (IL-1beta) and IL18, in osteoblasts. Cultured mouse osteoblasts were found to respond rapidly to either bacterial challenge by upregulation in the levels of mRNA encoding both IL-1beta and IL-18. Surprisingly, this mRNA expression did not translate into intracellular accumulation of IL-1beta or IL-18 precursor proteins or secretion of mature cytokines, despite the presence of detectable caspase-1 activity in these cells. These studies demonstrate that although osteoblasts can secrete a number of key proinflammatory mediators in response to bacterial pathogens, IL-1beta and IL-18 are not among this number. We suggest that osteoblasts are an unlikely source of these cytokines during the progression of bacterial infection of bone.

  5. Expression of LL-37, Human beta Defensin-2, and CCR6 mRNA in Patients with Psoriasis Vulgaris

    Institute of Scientific and Technical Information of China (English)

    李东升; 李家文; 段逸群; 周小勇

    2004-01-01

    To investigate whether LL-37 and human beta defensin-2 (hBD-2) is related to the patients with psoriasis seldom having skin infections and explore the role of the two peptides and CCR6 (the receptor of hBD-2) in the pathogenesis of psoriasis, the expression levels of mRNA of LL-37, hBD-2, and CCR6 in skin lesions of patients with psoriasis vulgaris were detected by using RT-PCR. The results showed that the mRNA expression levels of the two peptides and CCR6 in psoriatic lesions all increased compared with the normal skin (P<0. 001). It was suggested that upregulated expression of LL-37 and hBD-2 might be the main reason that result in the the skin of patients with psoriasis being seldom infected, and the two peptides and CCR6 might play crucial roles in the pathogenesis of psoriasis.

  6. Requirement of catalytically active Tyk2 and accessory signals for the induction of TRAIL mRNA by IFN-beta.

    Science.gov (United States)

    Rani, M R Sandhya; Pandalai, Sudha; Shrock, Jennifer; Almasan, Alex; Ransohoff, Richard M

    2007-09-01

    Tumor necrosis factor (TNF)-related apoptosis-inducing ligand/Apo2 ligand (TRAIL/Apo2L) mRNA was induced preferentially by interferon (IFN)-beta but not IFN-alpha in human fibrosarcoma and primary fibroblast cells. To characterize the signaling components mediating the IFN subtype-specific induction of this gene, we used mutant cell lines lacking individual components involved in signaling by type I IFNs. TRAIL was not induced by IFN-beta in mutant cell lines U2A, U3A, U4A, U5A, and U6A, which lack, respectively, IFN regulatory factor-9 (IRF-9), Stat1, Jak1, IFNAR-2.2, and Stat2, indicating transcription factor IFN-stimulated gene factor 3 (ISGF3) was essential for the induction of this gene. TRAIL was not induced by IFN-beta in U1A (Tyk2 null) or U1A.R930 cells (that express a kinase-deficient point mutant of Tyk2) but was induced in U1A.wt-5 cells (U1A cells expressing wild-type Tyk2), indicating that Tyk2 protein and kinase activity were both required for induction of the gene. Biochemical and genetic analyses revealed the requirement of transcription factor NF-kappa B and phosphoinositide 3-kinase (PI3K) but not extracellular signal-regulated kinase (ERK) for the induction of TRAIL by IFN-beta. Furthermore, the antiproliferative but not antiviral effects of IFN-beta required catalytically active Tyk2, suggesting that expression of genes, such as TRAIL, may play an important role in mediating the biologic effects of IFNs.

  7. Distribution of the mRNA for protein phosphatase T in rat brain

    NARCIS (Netherlands)

    Becker, W; Buttini, M; Limonta, S; Boddeke, H; Joost, HG

    1996-01-01

    We have recently cloned a novel protein serine/threonine phosphatase (PPT) from rat mRNA which is predominantly expressed in the brain (Becker et al., J. Biol. Chem., 269 (1994) 22586-22592). In the present study, the regional distribution of PPT mRNA in the brain of adult rats was characterized by

  8. Distribution of the mRNA for protein phosphatase T in rat brain

    NARCIS (Netherlands)

    Becker, W; Buttini, M; Limonta, S; Boddeke, H; Joost, HG

    1996-01-01

    We have recently cloned a novel protein serine/threonine phosphatase (PPT) from rat mRNA which is predominantly expressed in the brain (Becker et al., J. Biol. Chem., 269 (1994) 22586-22592). In the present study, the regional distribution of PPT mRNA in the brain of adult rats was characterized by

  9. Simultaneous isolation of mRNA and native protein from minute samples of cells

    DEFF Research Database (Denmark)

    Petersen, Tonny Studsgaard; Andersen, Claus Yding

    2014-01-01

    Precious biological samples often lack a sufficient number of cells for multiple procedures, such as extraction of mRNA while maintaining protein in a non-denatured state suitable for subsequent characterization. Here we present a new method for the simultaneous purification of mRNA and native pr...

  10. Selective estrogen receptor-beta (SERM-beta) compounds modulate raphe nuclei tryptophan hydroxylase-1 (TPH-1) mRNA expression and cause antidepressant-like effects in the forced swim test.

    Science.gov (United States)

    Clark, J A; Alves, S; Gundlah, C; Rocha, B; Birzin, E T; Cai, S-J; Flick, R; Hayes, E; Ho, K; Warrier, S; Pai, L; Yudkovitz, J; Fleischer, R; Colwell, L; Li, S; Wilkinson, H; Schaeffer, J; Wilkening, R; Mattingly, E; Hammond, M; Rohrer, S P

    2012-11-01

    Estrogen acts through two molecularly distinct receptors termed estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) which bind estradiol with similar affinities and mediate the effects of estrogen throughout the body. ERα plays a major role in reproductive physiology and behavior, and mediates classic estrogen signaling in such tissues as the uterus, mammary gland, and skeleton. ERβ, however, modulates estrogen signaling in the ovary, the immune system, prostate, gastrointestinal tract, and hypothalamus, and there is some evidence that ERβ can regulate ERα activity. Moreover, ERβ knockout studies and receptor distribution analyses in the CNS suggest that this receptor may play a role in the modulation of mood and cognition. In recent years several ERβ-specific compounds (selective estrogen receptor beta modulators; SERM-beta) have become available, and research suggests potential utility of these compounds in menopausal symptom relief, breast cancer prevention, diseases that have an inflammatory component, osteoporosis, cardiovascular disease, and inflammatory bowel disease, as well as modulation of mood, and anxiety. Here we demonstrate an antidepressant-like effect obtained using two SERM-beta compounds, SERM-beta1 and SERM-beta2. These compounds exhibit full agonist activity at ERβ in a cell based estrogen response element (ERE) transactivation assay. SERM-beta1 and 2 are non-proliferative with respect to breast as determined using the MCF-7 breast cancer cell-based assay and non-proliferative in the uterus as determined by assessing the effects of SERM-beta compounds on immature rat uterine weight and murine uterine weight. In vivo SERM-beta1 and 2 are brain penetrant and display dose dependent efficacy in the murine dorsal raphe assays for induction of tryptophan hydroxylase mRNA and progesterone receptor protein. These compounds show activity in the murine forced swim test and promote hippocampal neurogenesis acutely in rats. Taken

  11. Connecting protein and mRNA burst distributions for stochastic models of gene expression

    CERN Document Server

    Elgart, Vlad; Fenley, Andrew T; Kulkarni, Rahul V

    2011-01-01

    The intrinsic stochasticity of gene expression can lead to large variability in protein levels for genetically identical cells. Such variability in protein levels can arise from infrequent synthesis of mRNAs which in turn give rise to bursts of protein expression. Protein expression occurring in bursts has indeed been observed experimentally and recent studies have also found evidence for transcriptional bursting, i.e. production of mRNAs in bursts. Given that there are distinct experimental techniques for quantifying the noise at different stages of gene expression, it is of interest to derive analytical results connecting experimental observations at different levels. In this work, we consider stochastic models of gene expression for which mRNA and protein production occurs in independent bursts. For such models, we derive analytical expressions connecting protein and mRNA burst distributions which show how the functional form of the mRNA burst distribution can be inferred from the protein burst distributio...

  12. RNA-Binding Proteins Revisited – The Emerging Arabidopsis mRNA Interactome

    KAUST Repository

    Köster, Tino

    2017-04-13

    RNA–protein interaction is an important checkpoint to tune gene expression at the RNA level. Global identification of proteins binding in vivo to mRNA has been possible through interactome capture – where proteins are fixed to target RNAs by UV crosslinking and purified through affinity capture of polyadenylated RNA. In Arabidopsis over 500 RNA-binding proteins (RBPs) enriched in UV-crosslinked samples have been identified. As in mammals and yeast, the mRNA interactomes came with a few surprises. For example, a plethora of the proteins caught on RNA had not previously been linked to RNA-mediated processes, for example proteins of intermediary metabolism. Thus, the studies provide unprecedented insights into the composition of the mRNA interactome, highlighting the complexity of RNA-mediated processes.

  13. Live Borrelia burgdorferi preferentially activate interleukin-1 beta gene expression and protein synthesis over the interleukin-1 receptor antagonist.

    Science.gov (United States)

    Miller, L C; Isa, S; Vannier, E; Georgilis, K; Steere, A C; Dinarello, C A

    1992-01-01

    Lyme arthritis is one of the few forms of chronic arthritis in which the cause is known with certainty. Because cytokines are thought to contribute to the pathogenesis of chronic arthritis, we investigated the effect of the Lyme disease spirochete, Borrelia burgdorferi, on the gene expression and synthesis of IL-1 beta and the IL-1 receptor antagonist (IL-1ra) in human peripheral blood mononuclear cells. Live B. burgdorferi induced fivefold more IL-1 beta than IL-1 alpha and sevenfold more IL-1 beta than IL-1ra; LPS or sonicated B. burgdorferi induced similar amounts of all three cytokines. This preferential induction of IL-1 beta was most dramatic in response to a low passage, virulent preparation of B. burgdorferi vs. three high passage avirulent strains. No difference in induction of IL-1ra was seen between these strains. The marked induction of IL-1 beta was partially diminished by heat-treatment and abrogated by sonication; IL-1ra was not affected. This suggested that a membrane component(s) accounted for the preferential induction of IL-1 beta. However, recombinant outer surface protein beta induced little IL-1 beta. By 4 h after stimulation, B. burgdorferi induced sixfold more IL-1 beta protein than LPS. In contrast to LPS-induced IL-1 beta mRNA which reached maximal accumulation after 3 h, B. burgdorferi-induced IL-1 beta mRNA showed biphasic elevations at 3 and 18 h. B. burgdorferi-induced IL-1ra mRNA peaked at 12 h, whereas LPS-induced IL-1ra mRNA peaked at 9 h. IL-1 beta synthesis increased in response to increasing numbers of spirochetes, whereas IL-1ra synthesis did not. The preferential induction by B. burgdorferi of IL-1 beta over IL-1ra is an example of excess agonist over antagonist synthesis induced by a microbial pathogen, and may contribute to the destructive lesion of Lyme arthritis. Images PMID:1387885

  14. Search for antisense copies of beta-globin mRNA in anemic mouse spleen

    Directory of Open Access Journals (Sweden)

    Taylor John M

    2001-03-01

    Full Text Available Abstract Background Previous studies by Volloch and coworkers have reported that during the expression of high levels of β-globin mRNA in the spleen of anemic mice, they could also detect small but significant levels of an antisense (AS globin RNA species, which they postulated might have somehow arisen by RNA-directed RNA synthesis. For two reasons we undertook to confirm and possibly extend these studies. First, previous studies in our lab have focussed on what is an unequivocal example of host RNA-directed RNA polymerase activity on the RNA genome of human hepatitis delta virus. Second, if AS globin species do exist they could in turn form double-stranded RNA species which might induce post-transcriptional gene silencing, a phenomenon somehow provoked in eukaryotic cells by AS RNA sequences. Results We reexamined critical aspects of the previous globin studies. We used intraperitoneal injections of phenylhydrazine to induce anemia in mice, as demonstrated by the appearance and ultimate disappearance of splenomegaly. While a 30-fold increase in globin mRNA was detected in the spleen, the relative amount of putative AS RNA could be no more than 0.004%. Conclusions Contrary to earlier reports, induction of a major increase in globin transcripts in the mouse spleen was not associated with a detectable level of antisense RNA to globin mRNA.

  15. Macrocyclic beta-sheet peptides that mimic protein quaternary structure through intermolecular beta-sheet interactions.

    Science.gov (United States)

    Khakshoor, Omid; Demeler, Borries; Nowick, James S

    2007-05-02

    This paper reports the design, synthesis, and characterization of a family of cyclic peptides that mimic protein quaternary structure through beta-sheet interactions. These peptides are 54-membered-ring macrocycles comprising an extended heptapeptide beta-strand, two Hao beta-strand mimics [JACS 2000, 122, 7654] joined by one additional alpha-amino acid, and two delta-linked ornithine beta-turn mimics [JACS 2003, 125, 876]. Peptide 3a, as the representative of these cyclic peptides, contains a heptapeptide sequence (TSFTYTS) adapted from the dimerization interface of protein NuG2 [PDB ID: 1mio]. 1H NMR studies of aqueous solutions of peptide 3a show a partially folded monomer in slow exchange with a strongly folded oligomer. NOE studies clearly show that the peptide self-associates through edge-to-edge beta-sheet dimerization. Pulsed-field gradient (PFG) NMR diffusion coefficient measurements and analytical ultracentrifugation (AUC) studies establish that the oligomer is a tetramer. Collectively, these experiments suggest a model in which cyclic peptide 3a oligomerizes to form a dimer of beta-sheet dimers. In this tetrameric beta-sheet sandwich, the macrocyclic peptide 3a is folded to form a beta-sheet, the beta-sheet is dimerized through edge-to-edge interactions, and this dimer is further dimerized through hydrophobic face-to-face interactions involving the Phe and Tyr groups. Further studies of peptides 3b-3n, which are homologues of peptide 3a with 1-6 variations in the heptapeptide sequence, elucidate the importance of the heptapeptide sequence in the folding and oligomerization of this family of cyclic peptides. Studies of peptides 3b-3g show that aromatic residues across from Hao improve folding of the peptide, while studies of peptides 3h-3n indicate that hydrophobic residues at positions R3 and R5 of the heptapeptide sequence are important in oligomerization.

  16. The actin binding cytoskeletal protein Moesin is involved in nuclear mRNA export.

    Science.gov (United States)

    Kristó, Ildikó; Bajusz, Csaba; Borsos, Barbara N; Pankotai, Tibor; Dopie, Joseph; Jankovics, Ferenc; Vartiainen, Maria K; Erdélyi, Miklós; Vilmos, Péter

    2017-10-01

    Current models imply that the evolutionarily conserved, actin-binding Ezrin-Radixin-Moesin (ERM) proteins perform their activities at the plasma membrane by anchoring membrane proteins to the cortical actin network. Here we show that beside its cytoplasmic functions, the single ERM protein of Drosophila, Moesin, has a novel role in the nucleus. The activation of transcription by heat shock or hormonal treatment increases the amount of nuclear Moesin, indicating biological function for the protein in the nucleus. The distribution of Moesin in the nucleus suggests a function in transcription and the depletion of mRNA export factors Nup98 or its interacting partner, Rae1, leads to the nuclear accumulation of Moesin, suggesting that the nuclear function of the protein is linked to mRNA export. Moesin localizes to mRNP particles through the interaction with the mRNA export factor PCID2 and knock down of Moesin leads to the accumulation of mRNA in the nucleus. Based on our results we propose that, beyond its well-known, manifold functions in the cytoplasm, the ERM protein of Drosophila is a new, functional component of the nucleus where it participates in mRNA export. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. UCP-2 and UCP-3 Proteins Are Differentially Regulated in Pancreatic Beta-Cells

    Science.gov (United States)

    Li, Yunfeng; Maedler, Kathrin; Shu, Luan; Haataja, Leena

    2008-01-01

    Background Increased uncoupling protein-2 (UCP-2) expression has been associated with impaired insulin secretion, whereas UCP-3 protein levels are decreased in the skeleton muscle of type-2 diabetic subjects. In the present studies we hypothesize an opposing effect of glucose on the regulation of UCP-2 and UCP-3 in pancreatic islets. Methodology Dominant negative UCP-2 and wild type UCP-3 adenoviruses were generated, and insulin release by transduced human islets was measured. UCP-2 and UCP-3 mRNA levels were determined using quantitative PCR. UCP-2 and UCP-3 protein expression was investigated in human islets cultured in the presence of different glucose concentrations. Human pancreatic sections were analyzed for subcellular localization of UCP-3 using immunohistochemistry. Principal Findings Dominant negative UCP-2 expression in human islets increased insulin secretion compared to control islets (p<0.05). UCP-3 mRNA is expressed in human islets, but the relative abundance of UCP-2 mRNA was 8.1-fold higher (p<0.05). Immunohistochemical analysis confirmed co-localization of UCP-3 protein with mitochondria in human beta-cells. UCP-2 protein expression in human islets was increased ∼2-fold after high glucose exposure, whereas UCP-3 protein expression was decreased by ∼40% (p<0.05). UCP-3 overexpression improved glucose-stimulated insulin secretion. Conclusions UCP-2 and UCP-3 may have distinct roles in regulating beta-cell function. Increased expression of UCP-2 and decreased expression of UCP-3 in humans with chronic hyperglycemia may contribute to impaired glucose-stimulated insulin secretion. These data imply that mechanisms that suppress UCP-2 or mechanisms that increase UCP-3 expression and/or function are potential therapeutic targets to offset defects of insulin secretion in humans with type-2 diabetes. PMID:18167556

  18. UCP-2 and UCP-3 proteins are differentially regulated in pancreatic beta-cells.

    Directory of Open Access Journals (Sweden)

    Yunfeng Li

    Full Text Available BACKGROUND: Increased uncoupling protein-2 (UCP-2 expression has been associated with impaired insulin secretion, whereas UCP-3 protein levels are decreased in the skeleton muscle of type-2 diabetic subjects. In the present studies we hypothesize an opposing effect of glucose on the regulation of UCP-2 and UCP-3 in pancreatic islets. METHODOLOGY: Dominant negative UCP-2 and wild type UCP-3 adenoviruses were generated, and insulin release by transduced human islets was measured. UCP-2 and UCP-3 mRNA levels were determined using quantitative PCR. UCP-2 and UCP-3 protein expression was investigated in human islets cultured in the presence of different glucose concentrations. Human pancreatic sections were analyzed for subcellular localization of UCP-3 using immunohistochemistry. PRINCIPAL FINDINGS: Dominant negative UCP-2 expression in human islets increased insulin secretion compared to control islets (p<0.05. UCP-3 mRNA is expressed in human islets, but the relative abundance of UCP-2 mRNA was 8.1-fold higher (p<0.05. Immunohistochemical analysis confirmed co-localization of UCP-3 protein with mitochondria in human beta-cells. UCP-2 protein expression in human islets was increased approximately 2-fold after high glucose exposure, whereas UCP-3 protein expression was decreased by approximately 40% (p<0.05. UCP-3 overexpression improved glucose-stimulated insulin secretion. CONCLUSIONS: UCP-2 and UCP-3 may have distinct roles in regulating beta-cell function. Increased expression of UCP-2 and decreased expression of UCP-3 in humans with chronic hyperglycemia may contribute to impaired glucose-stimulated insulin secretion. These data imply that mechanisms that suppress UCP-2 or mechanisms that increase UCP-3 expression and/or function are potential therapeutic targets to offset defects of insulin secretion in humans with type-2 diabetes.

  19. Localized mRNA translation and protein association

    Science.gov (United States)

    Zhdanov, Vladimir

    2014-08-01

    Recent direct observations of localization of mRNAs and proteins both in prokaryotic and eukaryotic cells can be related to slowdown of diffusion of these species due to macromolecular crowding and their ability to aggregate and form immobile or slowly mobile complexes. Here, a generic kinetic model describing both these factors is presented and comprehensively analyzed. Although the model is non-linear, an accurate self-consistent analytical solution of the corresponding reaction-diffusion equation has been constructed, the types of localized protein distributions have been explicitly shown, and the predicted kinetic regimes of gene expression have been classified.

  20. A novel RNA binding protein that interacts with NMDA R1 mRNA: regulation by ethanol.

    Science.gov (United States)

    Anji, Antje; Kumari, Meena

    2006-05-01

    Excitatory NMDA receptors are an important target of ethanol. Chronic ethanol exposure, in vivo and in vitro, increases polypeptide levels of NR1 subunit, the key subunit of functional NMDA receptors. In vitro, chronic ethanol treatment increases the half-life of NR1 mRNA and this observation is dependent on new protein synthesis. The present study was undertaken to locate cis-acting region(s) within the NR1 3'-untranslated region (UTR) and identify NR1 3'-UTR binding trans-acting proteins expressed in mouse fetal cortical neurons. Utilizing RNA gel shift assays we identified a 156-nt cis-acting region that binds to polysomal trans-acting proteins. This binding was highly specific as inclusion of cyclophilin RNA or tRNA did not interfere with cis-trans interactions. Importantly, the 3'-UTR binding activity was significantly up-regulated in the presence of ethanol. UV cross-link analysis detected three NR1 3'-UTR binding proteins and their molecular mass calculated by Northwestern analysis was approximately 88, 60 and 47 kDa, respectively. Northwestern analysis showed a significant up-regulation of the 88-kDa protein after chronic ethanol treatment. The 88-kDa protein was purified and identified by tandem mass spectrometry as the beta subunit of alpha glucosidase II (GIIbeta). That GIIbeta is indeed a trans-acting protein and binds specifically to 3'-UTR of NR1 mRNA was confirmed by RNA gel mobility supershift assays and immuno RT-PCR. Western blotting data established a significant increase of GIIbeta polypeptide in chronic ethanol-exposed fetal cortical neurons. We hypothesize that the identified cis-acting region and the associated RNA-binding proteins are important regulators of NR1 subunit gene expression.

  1. Hyperresponsive febrile reactions to interleukin (IL) 1alpha and IL-1beta, and altered brain cytokine mRNA and serum cytokine levels, in IL-1beta-deficient mice.

    Science.gov (United States)

    Alheim, K; Chai, Z; Fantuzzi, G; Hasanvan, H; Malinowsky, D; Di Santo, E; Ghezzi, P; Dinarello, C A; Bartfai, T

    1997-03-18

    IL-1beta is an endogenous pyrogen that is induced during systemic lipopolysaccharide (LPS)- or IL-1-induced fever. We have examined the fever and cytokine responses following i.p. injection of IL-1 agonists, IL-1alpha and IL-1beta, and compared these with response to LPS (i.p.) in wild-type and IL-1beta-deficient mice. The IL-1beta deficient mice appear to have elevated body temperature but exhibit a normal circadian temperature cycle. Exogenously injected IL-1beta, IL-1alpha, or LPS induced hyperresponsive fevers in the IL-1beta-deficient mice. We also observed phenotypic differences between wild-type and IL-1beta-deficient mice in hypothalamic basal mRNA levels for IL-1alpha and IL-6, but not for IL-1beta-converting enzyme or IL-1 receptor type I or type II. The IL-1alpha mRNA levels were down-regulated, whereas the IL-6 mRNA levels were up-regulated in the hypothalamus of IL-1beta-deficient mice as compared with wild-type mice. The IL-1beta-deficient mice also responded to LPS challenge with significantly higher serum corticosterone and with lower serum tumor necrosis factor type alpha levels than the wild-type mice. The data suggest that, in the redundant cascade of proinflammatory cytokines, IL-1beta plays an important but not obligatory role in fever induction by LPS or IL-1alpha, as well as in the induction of serum tumor necrosis factor type alpha and corticosterone responses either by LPS or by IL-1alpha or IL-1beta.

  2. Role of the C terminus of Lassa virus L protein in viral mRNA synthesis.

    Science.gov (United States)

    Lehmann, Maria; Pahlmann, Meike; Jérôme, Hanna; Busch, Carola; Lelke, Michaela; Günther, Stephan

    2014-08-01

    The N terminus of arenavirus L protein contains an endonuclease presumably involved in "cap snatching." Here, we employed the Lassa virus replicon system to map other L protein sites that might be involved in this mechanism. Residues Phe-1979, Arg-2018, Phe-2071, Asp-2106, Trp-2173, Tyr-2179, Arg-2200, and Arg-2204 were important for viral mRNA synthesis but dispensable for genome replication. Thus, the C terminus of L protein is involved in the mRNA synthesis process, potentially by mediating cap binding.

  3. Tuning protein expression using synonymous codon libraries targeted to the 5' mRNA coding region

    DEFF Research Database (Denmark)

    Goltermann, Lise; Borch Jensen, Martin; Bentin, Thomas

    2011-01-01

    In bacteria, the 5' mRNA coding region plays an important role in determining translation output. Here, we report synthetic sequences that when placed in the 5'-mRNA coding region, leading to recombinant proteins containing short N-terminal extensions, virtually abolish, enhance or produce...... intermediate expression levels of green fluorescent protein in Escherichia coli. At least in one case, no apparent effect on protein stability was observed, pointing to RNA level effects as the principal reason for the observed expression differences. Targeting a synonymous codon library to the 5' coding...... and hence is important to recombinant and, most certainly, endogenous gene expression....

  4. Mutation of genes controlling mRNA metabolism and protein synthesis predisposes to neurodevelopmental disorders.

    Science.gov (United States)

    Sartor, Francesca; Anderson, Jihan; McCaig, Colin; Miedzybrodzka, Zosia; Müller, Berndt

    2015-12-01

    Brain development is a tightly controlled process that depends upon differentiation and function of neurons to allow for the formation of functional neural networks. Mutation of genes encoding structural proteins is well recognized as causal for neurodevelopmental disorders (NDDs). Recent studies have shown that aberrant gene expression can also lead to disorders of neural development. Here we summarize recent evidence implicating in the aetiology of NDDs mutation of factors acting at the level of mRNA splicing, mRNA nuclear export, translation and mRNA degradation. This highlights the importance of these fundamental processes for human health and affords new strategies and targets for therapeutic intervention.

  5. Presence of. beta. hCG mRNA in the human pituitary

    Energy Technology Data Exchange (ETDEWEB)

    van Strien, A.; van Wezenbeek, P. (Organon International BV, Oss (Netherlands))

    1989-07-11

    A strong structural and biological similarity exist between human chorionic gonadotropin (hCG) and luteinizing hormone (LH). They share the same {alpha} subunit and have evolutionary related {beta} subunits. hCG is synthesized in first trimester placenta, whereas LH is synthesized in the pituitary. Substances with immunological, physical and biological properties of hCG have, however, been observed in postmenopausal urinary gonadotropin preparations as well as in pituitary extracts. Despite this finding it has not yet been proven that the hCG-like material is identical to placental hCG. This report describes the presence of a minor messenger RNA species in the human pituitary. RNA was isolated from five human pituitary glands and an amplified cDNA library was prepared.

  6. Roles of Puf proteins in mRNA degradation and translation.

    Science.gov (United States)

    Miller, Melanie A; Olivas, Wendy M

    2011-01-01

    Puf proteins are regulators of diverse eukaryotic processes including stem cell maintenance, organelle biogenesis, oogenesis, neuron function, and memory formation. At the molecular level, Puf proteins promote translational repression and/or degradation of target mRNAs by first interacting with conserved cis-elements in the 3' untranslated region (UTR). Once bound to an mRNA, Puf proteins elicit RNA repression by complex interactions with protein cofactors and regulatory machinery involved in translation and degradation. Recent work has dramatically increased our understanding of the targets of Puf protein regulation, as well as the mechanisms by which Puf proteins recognize and regulate those mRNA targets. Crystal structure analysis of several Puf-RNA complexes has demonstrated that while Puf proteins are extremely conserved in their RNA-binding domains, Pufs attain target specificity by utilizing different structural conformations to recognize 8-10 nt sequences. Puf proteins have also evolved modes of protein interactions that are organism and transcript-specific, yet two common mechanisms of repression have emerged: inhibition of cap-binding events to block translation initiation, and recruitment of the CCR4-POP2-NOT deadenylase complex for poly(A) tail removal. Finally, multiple schemes to regulate Puf protein activity have been identified, including post-translational mechanisms that allow rapid changes in the repression of mRNA targets.

  7. Complement activation by the amyloid proteins A beta peptide and beta 2-microglobulin

    DEFF Research Database (Denmark)

    Nybo, Mads; Nielsen, E H; Svehag, S E

    1999-01-01

    Complement activation (CA) has been reported to play a role in the pathogenesis of Alzheimer's disease (AD). To investigate whether CA may contribute to amyloidogenesis in general, the CA potential of different amyloid fibril proteins was tested. CA induced by A beta preparations containing solub...

  8. Cellular differentiation state modulates the mRNA export activity of SR proteins.

    Science.gov (United States)

    Botti, Valentina; McNicoll, François; Steiner, Michaela C; Richter, Florian M; Solovyeva, Anfisa; Wegener, Marius; Schwich, Oliver D; Poser, Ina; Zarnack, Kathi; Wittig, Ilka; Neugebauer, Karla M; Müller-McNicoll, Michaela

    2017-07-03

    SR proteins function in nuclear pre-mRNA processing, mRNA export, and translation. To investigate their cellular dynamics, we developed a quantitative assay, which detects differences in nucleocytoplasmic shuttling among seven canonical SR protein family members. As expected, SRSF2 and SRSF5 shuttle poorly in HeLa cells but surprisingly display considerable shuttling in pluripotent murine P19 cells. Combining individual-resolution cross-linking and immunoprecipitation (iCLIP) and mass spectrometry, we show that elevated arginine methylation of SRSF5 and lower phosphorylation levels of cobound SRSF2 enhance shuttling of SRSF5 in P19 cells by modulating protein-protein and protein-RNA interactions. Moreover, SRSF5 is bound to pluripotency-specific transcripts such as Lin28a and Pou5f1/Oct4 in the cytoplasm. SRSF5 depletion reduces and overexpression increases their cytoplasmic mRNA levels, suggesting that enhanced mRNA export by SRSF5 is required for the expression of pluripotency factors. Remarkably, neural differentiation of P19 cells leads to dramatically reduced SRSF5 shuttling. Our findings indicate that posttranslational modification of SR proteins underlies the regulation of their mRNA export activities and distinguishes pluripotent from differentiated cells. © 2017 Botti et al.

  9. Expression of a novel beta adaptin subunit mRNA splice variant in human testes

    Institute of Scientific and Technical Information of China (English)

    Xin-Dong Zhang; Lan-Lan Yin; Ying Zheng; Li Lu; Zuo-Min Zhou; Jia-Hao Sha

    2005-01-01

    Aim: To identify a novel isoform of adaptin 2 beta subunit (named Ap2β-NY) and to investigate its relationship with testicular development and spermatogenesis. Methods: Using a human testis cDNA microarray, a clone (Ap2β-NY),which was strongly expressed in adult testes but weakly expressed in embryo testes, was sequenced and analyzed.Using polymerase chain reaction (PCR), the tissue distribution and expression time pattern of Ap2β-NY were determined.Results: Ap2β-NY was identified and has been deposited in the GenBank (AY341427). The expression level of Ap2β-NY in the adult testis was about 3-fold higher than that in the embryo testis. PCR analysis using multi-tissue cDNA indicated that Ap2β-NY was highly expressed in the testis, spleen, thymus, prostate, ovary, blood leukocyte and brain, but not in the heart, placenta, lung, liver, skeletal muscle, kidney and pancreas. In addition, Ap2β-NY was variably expressed in the testes of patients with spermatogenesis-disturbance and spermatogenesis-arrest but not expressed in those of Sertoli-cell-only syndrome, which implied that, in the testis, Ap2β-NY was restrictively expressed in germ cells. Conclusion: Ap2β-NY is an isoform of Ap2β and may be involved in regulating the process of spermatogenesis and testis development.

  10. O-GlcNAc-selective-N-acetyl-beta-D-glucosaminidase activity and mRNA expression in muscle is related to glucosamine-induced insulin resistance.

    Science.gov (United States)

    Durán-Reyes, Genoveva; Pascoe-Lira, Dalila; García-Macedo, Rebeca; Medina-Navarro, Rafael; Rosales-Torres, Ana María; Vergara-Onofre, Marcela; Foyo-Niembro, Enrique; Gutiérrez-Rodríguez, Margarita Eugenia; García-Gutiérrez, María Trinidad Adriana; Valladares-Salgado, Adán; Kumate, Jesús; Cruz, Miguel

    2010-01-01

    Glucosamine (GlcN)-induced insulin resistance is associated with an increase in O-linked-N-acetylglucosaminylated modified proteins (O-GlcNAcylated proteins). The role played by O-GlcNAc-selective-N-acetyl-beta-D-glucosaminidase (O-GlcNAcase), which removes O-N-acetyl-glucosamine residues from O-GlcNAcylated proteins, has not yet been demonstrated. We investigated whether GlcN-induced whole-body insulin resistance is related to tissue O-GlcNAcase activity and mRNA expression. GlcN (30 mumol/kg/min) or physiological saline (control) was intravenously infused into Sprague-Dawley rats for 2 h. After GlcN treatment, rats were subjected to the following: intravenous glucose tolerance test, insulin tolerance test or removal of the liver, muscle and pancreas. GlcN was found to provoke hyperglycemia compared to control (8.6 +/- 0.41 vs. 4.82 +/- 0.17 mM, p < 0.001). The insulin resistance index (HOMA-IR) increased (15.76 +/- 1.47 vs. 10.14 +/- 1.41, p < 0.001) and the beta-cell function index (HOMA-beta) diminished (182.69 +/- 22.37 vs. 592.01 +/- 103, p < 0.001). Liver glucose concentration was higher in the GlcN group than in the control group (0.37 +/- 0.04 vs. 0.24 +/- 0.038 mmol/g dry weight, p < 0.001). Insulin release index (insulin/glucose) was less in the GlcN group than in the control (2.2 +/- 0.1 vs. 8 +/- 0.8 at 120 min, p < 0.001). In the GlcN group, muscle O-GlcNAcase activity diminished (0.28 +/- 0.019 vs. 0.36 +/- 0.018 nmol of p-nitrophenyl/mg protein/min, p < 0.001), and K(m) increased (1.51 +/- 0.11 vs. 1.12 +/- 0.1 mM, p < 0.001) compared to the control. In the GlcN group, O-GlcNAcase activity/mRNA expression was altered (0.6 +/- 0.07 vs. 1 +/- 0.09 of control, p < 0.05). In conclusion, O-GlcNAcase activity is posttranslationally inhibited during GlcN-induced insulin resistance. Copyright (c) 2010 S. Karger AG, Basel.

  11. Cytokines in relapsing experimental autoimmune encephalomyelitis in DA rats: persistent mRNA expression of proinflammatory cytokines and absent expression of interleukin-10 and transforming growth factor-beta.

    Science.gov (United States)

    Issazadeh, S; Lorentzen, J C; Mustafa, M I; Höjeberg, B; Müssener, A; Olsson, T

    1996-09-01

    Experimental autoimmune encephalomyelitis (EAE) in rats is typically a brief and monophasic disease with sparse demyelination. However, inbred DA rats develop a demyelinating, prolonged and relapsing encephalomyelitis after immunization with rat spinal cord in incomplete Freund's adjuvant. This model enables studies of mechanisms related to chronicity and demyelination, two hallmarks of multiple sclerosis (MS). Here we have investigated, in situ, the dynamics of cytokine mRNA expression in the central nervous system (CNS) and peripheral lymphoid organs (lymph node cells and splenocytes) of diseased DA rats. We demonstrate that peripheral lymphoid cells stimulated in vitro with encephalitogenic peptides 69-87 and 87-101 of myelin basic protein responded with high mRNA expression for proinflammatory cytokines; interferon-gamma, interleukin-12 (IL-12), tumour necrosis factors alpha and beta, IL-1 beta and cytolysin. A high expression of mRNA for these proinflammatory cytokines was also observed in the CNS where it was accompanied by classical signs of inflammation such as expression of major histocompatibility complex class I and II, CD4, CD8 and IL-2 receptor. The expression of mRNA for proinflammatory cytokines was remarkably long-lasting in DA rats as compared to LEW rats which display a brief and monophasic EAE. Furthermore, mRNAs for putative immunodownmodulatory cytokines, i.e. transforming growth factor-beta (TGF-beta), IL-10 and IL-4 were almost absent in DA rats, in both the CNS and in vitro stimulated peripheral lymphoid cells, while their levels were elevated in the CNS of LEW rats during the recovery phase. We conclude that the MS-like prolonged and relapsing EAE in DA rats is associated with a prolonged production of proinflammatory cytokines and/or low or absent production of immunodownmodulatory cytokines.

  12. Myxovirus Resistance Protein A mRNA Expression Kinetics in Multiple Sclerosis Patients Treated with IFNβ

    Science.gov (United States)

    Libertinova, Jana; Meluzinova, Eva; Tomek, Ales; Horakova, Dana; Kovarova, Ivana; Matoska, Vaclav; Kumstyrova, Simona; Zajac, Miroslav; Hyncicova, Eva; Liskova, Petra; Houzvickova, Eva; Martinkovic, Lukas; Bojar, Martin; Havrdova, Eva; Marusic, Petr

    2017-01-01

    Introduction Interferon-β (IFNß) is the first-line treatment for relapsing-remitting multiple sclerosis. Myxovirus resistance protein A (MxA) is a marker of IFNß bioactivity, which may be reduced by neutralizing antibodies (NAbs) against IFNß. The aim of the study was to analyze the kinetics of MxA mRNA expression during long-term IFNβ treatment and assess its predictive value. Methods A prospective, observational, open-label, non-randomized study was designed in multiple sclerosis patients starting IFNß treatment. MxA mRNA was assessed prior to initiation of IFNß therapy and every three months subsequently. NAbs were assessed every six months. Assessment of relapses was scheduled every three months during 24 months of follow up. The disease activity was correlated to the pretreatment baseline MxA mRNA value. In NAb negative patients, clinical status was correlated to MxA mRNA values. Results 119 patients were consecutively enrolled and 107 were included in the final analysis. There was no correlation of MxA mRNA expression levels between baseline and month three. Using survival analysis, none of the selected baseline MxA mRNA cut off points allowed prediction of time to first relapse on the treatment. In NAb negative patients, mean MxA mRNA levels did not significantly differ in patients irrespective of relapse status. Conclusion Baseline MxA mRNA does not predict the response to IFNß treatment or the clinical status of the disease and the level of MxA mRNA does not correlate with disease activity in NAb negative patients. PMID:28081207

  13. Measurement of MxA mRNA or protein as a biomarker of IFNbeta bioactivity: detection of antibody-mediated decreased bioactivity (ADB).

    Science.gov (United States)

    Pachner, Andrew R; Bertolotto, Antonio; Deisenhammer, Florian

    2003-11-11

    Myxovirus A (MxA) is a protein that is specifically induced by treatment with type I cytokines and has proven to be a reliable biomarker of interferon-beta (IFNbeta) bioactivity. IFNbeta-induced MxA can be measured as either protein or mRNA in the blood of IFNbeta-treated patients with MS. In patients with MS who are treated with IFNbeta, loss of the MxA response is caused by high levels of anti-IFNbeta antibodies and is a sensitive marker of lost bioactivity.

  14. Epithelial sodium channel (ENaC) subunit mRNA and protein expression in rats with puromycin aminonucleoside-induced nephrotic syndrome.

    Science.gov (United States)

    Audigé, A; Yu, Z R; Frey, B M; Uehlinger, D E; Frey, F J; Vogt, B

    2003-04-01

    In experimental nephrotic syndrome, urinary sodium excretion is decreased during the early phase of the disease. The molecular mechanism(s) leading to salt retention has not been completely elucidated. The rate-limiting constituent of collecting duct sodium transport is the epithelial sodium channel (ENaC). We examined the abundance of ENaC subunit mRNAs and proteins in puromycin aminonucleoside (PAN)-induced nephrotic syndrome. The time courses of urinary sodium excretion, plasma aldosterone concentration and proteinuria were studied in male Sprague-Dawley rats treated with a single dose of either PAN or vehicle. The relative amounts of alphaENaC, betaENaC and gammaENaC mRNAs were determined in kidneys from these rats by real-time quantitative TaqMan PCR, and the amounts of proteins by Western blot. The kinetics of urinary sodium excretion and the appearance of proteinuria were comparable with those reported previously. Sodium retention occurred on days 2, 3 and 6 after PAN injection. A significant up-regulation of alphaENaC and betaENaC mRNA abundance on days 1 and 2 preceded sodium retention on days 2 and 3. Conversely, down-regulation of alphaENaC, betaENaC and gammaENaC mRNA expression on day 3 occurred in the presence of high aldosterone concentrations, and was followed by a return of sodium excretion to control values. The amounts of alphaENaC, betaENaC and gammaENaC proteins were not increased during PAN-induced sodium retention. In conclusion, ENaC mRNA expression, especially alphaENaC, is increased in the very early phase of the experimental model of PAN-induced nephrotic syndrome in rats, but appears to escape from the regulation by aldosterone after day 3.

  15. [Interrelationship between protein synthesis and mRNA metabolism in rat liver cells].

    Science.gov (United States)

    Arbuzov, V A

    1976-08-01

    It is demonstrated that RNA isolated from polyribosomes and postmitochondrial fraction of rat liver cells and bound to nitrocellulose filters (Milliport) represent mRNA. RNA taken from the nitrocellulose filters sedimented in sucrose concentration gradient with a wide peak within the range of 18--6S, attaining a maximum at 12S. The (A+U)/(G+C) ratio of this RNA was equal to 1.04. On the other hand, the same ratio for rRNA was 0.64. Specific radioactivity of polysomal mRNA containing poly-A sequences, was significantly lower at 14-hour labelling with 14C-orotate than at 4-hour labelling (control). Inhibitors (cycloheximide, puromycin, ethionine, actinomycin D) stabilized polysomal mRNA. Specific radioactivity of postmitochondrial fraction mRNA was higher at 14-hour labelling than at 4-hour labelling. Specific radioactivity of postmitochondrial fraction mRNA during protein synthesis blocking by different inhibitors was comparable to those of control animals. It is hypothesized that active translation is necessary for the initiation of rat liver mRNA degradation.

  16. Effects of irradiation on TGF-{beta}{sub 1} mRNA expression and calcific nodule formation in MC3T3-E1 osteoblastic cell line

    Energy Technology Data Exchange (ETDEWEB)

    Song, Ju Seop; Kim, Kyoung A; Koh, Kwang Joon [Department of Oral and Maxillofacial Radiology, School of Dentistry, and Institute of Oral Bio Science, Chonbuk National University, Jeonju (Korea, Republic of)

    2008-09-15

    To investigate the effects of irradiation on transforming growth factor {beta}1 (TGF-{beta}{sub 1}) mRNA expression and calcific nodule formation in MC3T3-E1 osteoblastic cell line. Cells were cultured in alpha-minimum essential medium ({alpha}-MEM) supplemented with 10% fetal bovine serum and antibiotics. When the cells reached the level of 70-80% confluence, culture media were changed with {alpha}-MEM supplemented with 10% FBS, 5 mM {beta}-glycerol phosphate, and 50 {mu}g/mL ascorbic acid. Thereafter the cells were irradiated with a single dose of 2, 4, 6, 8 Gy at a dose rate of 1.5 Gy/min. The expression pattern of TGF-{beta}{sub 1} mRNA, calcium content and calcific nodule formation were examined on day 3, 7, 14, 21, 28, respectively, after the irradiation. The amount of TGF-{beta}{sub 1} mRNA expression decreased significantly on day 7 after irradiation of 4, 6, 8 Gy. It also decreased on day 14 after irradiation of 6, 8 Gy, and decreased on day 21 after irradiation of 8 Gy. The amount of calcium deposition decreased significantly on day 7 after irradiation of 4, 8 Gy (P<0.01) and showed a decreased tendency on day 14, 21 after irradiation of 4, 6, 8 Gy. The number of calcific nodules was decreased on day 7 after irradiation of 4, 8 Gy. Irradiation with a single dose of 4, 6, 8 Gy influences negatively the bone formation at the molecular level by affecting the TGF-{beta}{sub 1} mRNA expression that was associated with proliferation and the production of extracellular matrix in MC3T3-E1 osteoblastic cell line

  17. Proprotein convertase 1 mRNA and protein expression in ischemic rat cortex after reperfusion

    Institute of Scientific and Technical Information of China (English)

    Shuqin Zhan; An Zhou; Jingquan Lan; Tao Yang

    2011-01-01

    Proprotein convertase 1 (PC1) is a member of the family of proprotein convertases (PCs), which are the processing enzymes of neuropeptides. Previous studies have addressed PC1 effects with regard to the neuroendocrine system. In this study, the developing changes of PC1 mRNA and PC1 protein in rat cortices after transient focal cerebral ischemia were investigated by fluorescent double labeling (both in situ hybridization and immunocytochemistry) using a transient focal cerebral ischemia model in rats. The results were compared with those of sham-operated rat cortices. Both the mRNA and protein levels of PC1 in ischemic cortices decreased gradually at 4, 8, and 16 hours of reperfusion after 100 minutes of middle cerebral artery occlusion. After 24 hours of reperfusion, enhanced intensities of signals for PC1 protein were observed, while signals for PC1 mRNA remained low. These results suggest that transient focal cerebral ischemia influences PC1 mRNA and protein expression in cortices of ischemic rats. Thus, PC1 is regulated by ischemic stress.

  18. Effect of taurine on mRNA expression of thioredoxin interacting protein in Caco-2 cells.

    Science.gov (United States)

    Gondo, Yusuke; Satsu, Hideo; Ishimoto, Yoko; Iwamoto, Taku; Shimizu, Makoto

    2012-09-28

    Taurine (2-aminoethanesulfonic acid), a sulfur-containing β-amino acid, plays an important role in several essential biological processes; although, the underlying mechanisms for these regulatory functions remain to be elucidated, especially at the genetic level. We investigated the effects of taurine on the gene expression profile in Caco-2 cells using DNA microarray. Taurine increased the mRNA expression of thioredoxin interacting protein (TXNIP), which is involved in various metabolisms and diseases. β-Alanine or γ-aminobutyric acid (GABA), which are structurally or functionally related to taurine, did not increase TXNIP mRNA expression. These suggest the expression of TXNIP mRNA is induced specifically by taurine. β-Alanine is also known to be a substrate of taurine transporter (TAUT) and competitively inhibits taurine uptake. Inhibition of taurine uptake by β-alanine eliminated the up-regulation of TXNIP, which suggests TAUT is involved in inducing TXNIP mRNA expression. The up-regulation of TXNIP mRNA expression by taurine was also observed at the protein level. Furthermore, taurine significantly increased TXNIP promoter activity. Our present study demonstrated the taurine-specific phenomenon of TXNIP up-regulation, which sheds light on the physiological function of taurine. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Proteins involved in the degradation of cytoplasmic mRNA in the major eukaryotic model systems.

    Science.gov (United States)

    Siwaszek, Aleksandra; Ukleja, Marta; Dziembowski, Andrzej

    2014-01-01

    The process of mRNA decay and surveillance is considered to be one of the main posttranscriptional gene expression regulation platforms in eukaryotes. The degradation of stable, protein-coding transcripts is normally initiated by removal of the poly(A) tail followed by 5'-cap hydrolysis and degradation of the remaining mRNA body by Xrn1. Alternatively, the exosome complex degrades mRNA in the 3'>5'direction. The newly discovered uridinylation-dependent pathway, which is present in many different organisms, also seems to play a role in bulk mRNA degradation. Simultaneously, to avoid the synthesis of incorrect proteins, special cellular machinery is responsible for the removal of faulty transcripts via nonsense-mediated, no-go, non-stop or non-functional 18S rRNA decay. This review is focused on the major eukaryotic cytoplasmic mRNA degradation pathways showing many similarities and pointing out main differences between the main model-species: yeast, Drosophila, plants and mammals.

  20. Selective translation of the measles virus nucleocapsid mRNA by La protein

    Directory of Open Access Journals (Sweden)

    Yoshihisa eInoue

    2011-08-01

    Full Text Available Measles, caused by measles virus (MeV infection, is the leading cause of death in children because of secondary infections attributable to MeV-induced immune suppression. Recently, we have shown that wild-type MeVs induce the suppression of protein synthesis in host cells (referred to as "shutoff" and that viral mRNAs are preferentially translated under shutoff conditions in infected cells. To determine the mechanism behind the preferential translation of viral mRNA, we focused on the 5 untranslated region (UTR of nucleocapsid (N mRNA. The La/SSB autoantigen (La was found to specifically bind to an N-5UTR probe. Recombinant La enhanced the translation of luciferase mRNA containing the N-5UTR (N-fLuc, and RNA interference of La suppressed N-fLuc translation. Furthermore, recombinant MeV lacking the La-binding motif in the N-5UTR displayed delayed viral protein synthesis and growth kinetics at an early phase of infection. These results suggest that La induced predominant translation of N mRNA via binding to its 5UTR under shutoff conditions. This is the first report on a cellular factor that specifically regulates paramyxovirus mRNA translation.

  1. Optimization of mRNA design for protein expression in the crustacean Daphnia magna.

    Science.gov (United States)

    Törner, Kerstin; Nakanishi, Takashi; Matsuura, Tomoaki; Kato, Yasuhiko; Watanabe, Hajime

    2014-08-01

    The water flea Daphnia is a new model organism for ecological, evolutionary, and toxicological genomics. Detailed functional analysis of genes newly discovered through genomic approaches often requires overexpression of the identified protein. In the present study, we report the microinjection of in vitro-synthesized RNAs into the eggs as a method for overexpressing ubiquitous proteins in Daphnia magna. We injected a 1.3-kb mRNA that coded for the red fluorescent protein (DsRed2) flanked by UTRs from the ubiquitously expressed elongation factor 1α-1 (EF1α-1) into D. magna embryos. DsRed2 fluorescence in the embryos was measured 24 h after microinjection. Unexpectedly, the reporter RNA containing the 522-bp full-length EF1α-1 3' UTR failed to induce fluorescence. To assess reporter expression, the length of the 3' UTR that potentially contained negative regulatory elements of protein expression, including AU-rich regions and Musashi binding elements, was serially reduced from the 3' end. Assessing all injected RNA alternatives, mRNA containing the first 60 bp of the 3' UTR gave rise to the highest fluorescence, 14 times the Daphnia auto-fluorescence. In contrast, mRNA lacking the entire 3' UTR hardly induced any change in fluorescence intensity. This is the first evaluation of UTRs of mRNAs delivered into Daphnia embryos by microinjection for overexpressing proteins. The mRNA with truncated 3' UTRs of Daphnia EF1α-1 will be useful not only for gain-of-function analyses but also for labeling proteins and organelles with fluorescent proteins in Daphnia.

  2. Synthetic mRNA devices that detect endogenous proteins and distinguish mammalian cells.

    Science.gov (United States)

    Kawasaki, Shunsuke; Fujita, Yoshihiko; Nagaike, Takashi; Tomita, Kozo; Saito, Hirohide

    2017-07-07

    Synthetic biology has great potential for future therapeutic applications including autonomous cell programming through the detection of protein signals and the production of desired outputs. Synthetic RNA devices are promising for this purpose. However, the number of available devices is limited due to the difficulty in the detection of endogenous proteins within a cell. Here, we show a strategy to construct synthetic mRNA devices that detect endogenous proteins in living cells, control translation and distinguish cell types. We engineered protein-binding aptamers that have increased stability in the secondary structures of their active conformation. The designed devices can efficiently respond to target proteins including human LIN28A and U1A proteins, while the original aptamers failed to do so. Moreover, mRNA delivery of an LIN28A-responsive device into human induced pluripotent stem cells (hiPSCs) revealed that we can distinguish living hiPSCs and differentiated cells by quantifying endogenous LIN28A protein expression level. Thus, our endogenous protein-driven RNA devices determine live-cell states and program mammalian cells based on intracellular protein information. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interactions and gene expression profiling

    Directory of Open Access Journals (Sweden)

    Mo Min

    2008-05-01

    Full Text Available Abstract Background The polyadenylation of mRNA is one of the critical processing steps during expression of almost all eukaryotic genes. It is tightly integrated with transcription, particularly its termination, as well as other RNA processing events, i.e. capping and splicing. The poly(A tail protects the mRNA from unregulated degradation, and it is required for nuclear export and translation initiation. In recent years, it has been demonstrated that the polyadenylation process is also involved in the regulation of gene expression. The polyadenylation process requires two components, the cis-elements on the mRNA and a group of protein factors that recognize the cis-elements and produce the poly(A tail. Here we report a comprehensive pairwise protein-protein interaction mapping and gene expression profiling of the mRNA polyadenylation protein machinery in Arabidopsis. Results By protein sequence homology search using human and yeast polyadenylation factors, we identified 28 proteins that may be components of Arabidopsis polyadenylation machinery. To elucidate the protein network and their functions, we first tested their protein-protein interaction profiles. Out of 320 pair-wise protein-protein interaction assays done using the yeast two-hybrid system, 56 (~17% showed positive interactions. 15 of these interactions were further tested, and all were confirmed by co-immunoprecipitation and/or in vitro co-purification. These interactions organize into three distinct hubs involving the Arabidopsis polyadenylation factors. These hubs are centered around AtCPSF100, AtCLPS, and AtFIPS. The first two are similar to complexes seen in mammals, while the third one stands out as unique to plants. When comparing the gene expression profiles extracted from publicly available microarray datasets, some of the polyadenylation related genes showed tissue-specific expression, suggestive of potential different polyadenylation complex configurations. Conclusion An

  4. Adaptive and maladaptive expression of the mRNA regulatory protein HuR

    Institute of Scientific and Technical Information of China (English)

    Suman; Govindaraju; Beth; S; Lee

    2013-01-01

    The RNA-binding proteins involved in regulation of mRNA post-transcriptional processing and translation control the fates of thousands of mRNA transcripts and basic cellular processes. The best studied of these, HuR, is well characterized as a mediator of mRNA stability and translation, and more recently, as a factor in nuclear functions such as pre-mRNA splicing. Due to HuR’s role in regulating thousands of mRNA transcripts, including those for other RNA-binding proteins, HuR can act as a master regulator of cell survival and proliferation. HuR itself is subject to multiple post-translationa modifications including regulation of its nucleocytoplasmic distribution. However, the mechanisms that govern HuR levels in the cell have only recently begun to be defined. These mechanisms are critical to cell health, as it has become clear in recent years that aberrant expression of HuR can lead alternately to decreased cell viability or to promotion of pathological proliferation and invasiveness. HuR is expressed as alternate mRNAs that vary in their untranslated regions, leading to differences in transcript stability and translatability. Multiple transcription factors and modulators of mRNA stability that regulate HuR mRNA expression have been identified. In addition, translation of HuR is regulated by numerous microRNAs, several of which have been demonstrated to have anti-tumor properties due to their suppression of HuR expression. This review summarizes the current state of knowledge of the factors that regulate HuR expression, along with the circumstances under which these factors contribute to cancer and inflammation.

  5. Regulation of the growth hormone (GH) receptor and GH-binding protein mRNA

    Energy Technology Data Exchange (ETDEWEB)

    Kaji, Hidesuke; Ohashi, Shin-Ichirou; Abe, Hiromi; Chihara, Kazuo [Kobe Univ. School of Medicine, Kobe (Japan)

    1994-12-31

    In fasting rats, a transient increase in growth hormone-binding protein (GHBP) mRNA levels was observed after 1 day, in muscle, heart, and liver, but not in fat tissues. The liver GH receptor (GHR) mRNA level was significantly increased after 1 day (but not after 5 days) of bovine GH (bGH) treatment in fed rats. Both the liver GHR mRNA level and the net increment of plasma IGF-I markedly decreased after 5 days of bGH administration in fasting rats. These findings suggest that GHR and GHBP mRNAs in the liver are expressed in a different way and that the expression of GHBP mRNA is regulated differently between tissues, at least in rats. The results also suggest that refractoriness to GH in a sustained fasting state might be beneficial in preventing anabolic effects of GH. In humans, GHR mRNA in lymphocytes, from subjects with either GH-deficiency or acromegaly, could be detected by the reverse transcription-polymerase chain reaction method. In one patient with partial GH insensitivity, a heterozygous missense mutation (P561T) was identified in the cytoplasmic domain of GHR. 15 refs., 4 figs.

  6. The Nuclear PolyA-Binding Protein Nab2p Is Essential for mRNA Production.

    Science.gov (United States)

    Schmid, Manfred; Olszewski, Pawel; Pelechano, Vicent; Gupta, Ishaan; Steinmetz, Lars M; Jensen, Torben Heick

    2015-07-07

    Polyadenylation of mRNA is a key step in eukaryotic gene expression. However, despite the major impact of poly(A) tails on mRNA metabolism, the precise roles of poly(A)-binding proteins (PABPs) in nuclear mRNA biogenesis remain elusive. Here, we demonstrate that rapid nuclear depletion of the S. cerevisiae PABP Nab2p leads to a global loss of cellular mRNA, but not of RNA lacking poly(A) tails. Disappearance of mRNA is a nuclear event, but not due to decreased transcription. Instead, the absence of Nab2p results in robust nuclear mRNA decay by the ribonucleolytic RNA exosome in a polyadenylation-dependent process. We conclude that Nab2p is required to protect early mRNA and therefore constitutes a crucial nuclear mRNA biogenesis factor. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  7. The Nuclear PolyA-Binding Protein Nab2p Is Essential for mRNA Production

    DEFF Research Database (Denmark)

    Schmid, Manfred; Olszewski, Pawel; Pelechano, Vicent;

    2015-01-01

    Polyadenylation of mRNA is a key step in eukaryotic gene expression. However, despite the major impact of poly(A) tails on mRNA metabolism, the precise roles of poly(A)-binding proteins (PABPs) in nuclear mRNA biogenesis remain elusive. Here, we demonstrate that rapid nuclear depletion of the S....... cerevisiae PABP Nab2p leads to a global loss of cellular mRNA, but not of RNA lacking poly(A) tails. Disappearance of mRNA is a nuclear event, but not due to decreased transcription. Instead, the absence of Nab2p results in robust nuclear mRNA decay by the ribonucleolytic RNA exosome in a polyadenylation......-dependent process. We conclude that Nab2p is required to protect early mRNA and therefore constitutes a crucial nuclear mRNA biogenesis factor....

  8. MHC2TA mRNA levels and human herpesvirus 6 in multiple sclerosis patients treated with interferon beta along two-year follow-up

    Directory of Open Access Journals (Sweden)

    Dominguez-Mozo Maria Inmaculada

    2012-09-01

    Full Text Available Abstract Background In previous studies we found that MHC2TA +1614 genotype frequency was very different when MS patients with and without human herpesvirus 6 (HHV-6 in serum samples were compared; a different clinical behavior was also described. The purpose of the study was: 1. To evaluate if MHC2TA expression in MS patients was influenced by interferon beta (IFN-beta treatment. 2. To study MHC2TA expression in MS patients with and without minor allele C. 3. To analyze the relation between MHC2TA mRNA levels and HHV-6 active infection in MS patients. Methods Blood and serum samples of 154 MS patients were collected in five programmed visits: basal (prior to beginning IFN-beta treatment, six, twelve, eighteen and twenty-four months later. HHV-6 in serum and MHC2TA mRNA levels were evaluated by PCR and RT-PCR, respectively. Neutralizing antibodies (NAbs against IFN-beta were analyzed by the cytopathic effect assay. Results We found that MHC2TA mRNA levels were significantly lower among MS patients with HHV-6 active infection at the basal visit (without treatment than in those MS patients without HHV-6 active infection at the basal visit (p = 0.012; in all the positive samples we only found variant A. Furthermore, 58/99 (58.6% MS patients without HHV-6 along the five programmed visits and an increase of MHC2TA expression after two-years of IFN-beta treatment were clinical responders vs. 5/21 (23.8% among those MS patients with HHV-6 and a decrease of MHC2TA mRNA levels along the two-years with IFN-beta treatment (p = 0.004; no differences were found between patients with and without NAbs. Conclusions MHC2TA mRNA levels could be decreased by the active replication of HHV-6; the absence of HHV-6 in serum and the increase of MHC2TA expression could be further studied as markers of good clinical response to IFN-beta treatment.

  9. Polynucleotide phosphorylase hinders mRNA degradation upon ribosomal protein S1 overexpression in Escherichia coli.

    Science.gov (United States)

    Briani, Federica; Curti, Serena; Rossi, Francesca; Carzaniga, Thomas; Mauri, Pierluigi; Dehò, Gianni

    2008-11-01

    The exoribonuclease polynucleotide phosphorylase (PNPase, encoded by pnp) is a major player in bacterial RNA decay. In Escherichia coli, PNPase expression is post-transcriptionally regulated at the level of mRNA stability. The primary transcript is very efficiently processed by the endonuclease RNase III at a specific site and the processed pnp mRNA is rapidly degraded in a PNPase-dependent manner. While investigating the PNPase autoregulation mechanism we found, by UV-cross-linking experiments, that the ribosomal protein S1 in crude extracts binds to the pnp-mRNA leader region. We assayed the potential role of S1 protein in pnp gene regulation by modulating S1 expression from depletion to overexpression. We found that S1 depletion led to a sharp decrease of the amount of pnp and other tested mRNAs, as detected by Northern blotting, whereas S1 overexpression caused a strong stabilization of pnp and the other transcripts. Surprisingly, mRNA stabilization depended on PNPase, as it was not observed in a pnp deletion strain. PNPase-dependent stabilization, however, was not detected by chemical decay assay of bulk mRNA. Overall, our data suggest that PNPase exonucleolytic activity may be modulated by the translation potential of the target mRNAs and that, upon ribosomal protein S1 overexpression, PNPase protects from degradation a set of full-length mRNAs. It thus appears that a single mRNA species may be differentially targeted to either decay or PNPase-dependent stabilization, thus preventing its depletion in conditions of fast turnover.

  10. Real time imaging of mRNA expression dynamics in live cells using protein complementation methods

    Science.gov (United States)

    Meller, Amit

    2009-03-01

    Traditional methods for mRNA quantification in cells, such as northern blots, quantitative PCR or microarrays assays, require cell lysis and therefore do not preserve its dynamics. These methods cannot be used to probe the spatio-temporal localization of mRNA in cells, which provide useful information for a wide range biomolecular process, including RNA metabolizim, expression kinetics and RNA interference. To probe mRNA dynamics in live prokaryotic and eukaryotic cells, we develop a method, which exploit the strong affinity of the eukaryotic initiation factor 4A (eIF4A) to specific RNA aptamers. Two parts of the eIF4A are fused to a split Green Fluorescence Protein (GFP), and are expressed in the cells at high abundance. However, only when the RNA apatmer is also present, the two protein parts complement and become fluorescent. Thus, the fluorescent background remains low, allowing us to directly image the expression of mRNA molecules in live e-coli cells from its early onset, over hours. We find that the expression kinetics can be classified in one out of at least three forms, which also display distinct spatial distributions. I will discuss the possible biological origin for these distributions and their time evolution.

  11. Making myelin basic protein -from mRNA transport to localized translation.

    Science.gov (United States)

    Müller, Christina; Bauer, Nina M; Schäfer, Isabelle; White, Robin

    2013-09-27

    In the central nervous system (CNS) of most vertebrates, oligodendrocytes enwrap neuronal axons with extensions of their plasma membrane to form the myelin sheath. Several proteins are characteristically found in myelin of which myelin basic protein (MBP) is the second most abundant one after proteolipid protein. The lack of functional MBP in rodents results in a severe hypomyelinated phenotype in the CNS demonstrating its importance for myelin synthesis. Mbp mRNA is transported from the nucleus to the plasma membrane and is translated locally at the axon-glial contact site. Axonal properties such as diameter or electrical activity influence the degree of myelination. As oligodendrocytes can myelinate many axonal segments with varying properties, localized MBP translation represents an important part of a rapid and axon-tailored synthesis machinery. MBP's ability to compact cellular membranes may be problematic for the integrity of intracellular membranous organelles and can also explain why MBP is transported in oligodendrocytes in the form of an mRNA rather than as a protein. Here we review the recent findings regarding intracellular transport and signaling mechanisms leading to localized translation of Mbp mRNA in oligodendrocytes. More detailed insights into the MBP synthesis pathway are important for a better understanding of the myelination process and may foster the development of remyelination therapies for demyelinating diseases.

  12. Making Myelin Basic Protein -from mRNA transport to localized translation

    Directory of Open Access Journals (Sweden)

    Christina eMüller

    2013-09-01

    Full Text Available In the central nervous system (CNS of most vertebrates, oligodendrocytes enwrap neuronal axons with extensions of their plasma membrane to form the myelin sheath. Several proteins are characteristically found in myelin of which Myelin Basic Protein (MBP is the second most abundant one after Proteolipid Protein (PLP. The lack of functional MBP in rodents results in a severe hypomyelinated phenotype in the CNS demonstrating its importance for myelin synthesis. Mbp mRNA is transported from the nucleus to the plasma membrane and is translated locally at the axon-glial contact site. Axonal properties such as diameter or electrical activity influence the degree of myelination. As oligodendrocytes can myelinate many axonal segments with varying properties, localized MBP translation represents an important part of a rapid and axon-tailored synthesis machinery. MBP’s ability to compact cellular membranes may be problematic for the integrity of intracellular membranous organelles and can also explain why MBP is transported in oligodendrocytes in the form of an mRNA rather than as a protein. Here we review the recent findings regarding intracellular transport and signalling mechanisms leading to localized translation of Mbp mRNA in oligodendrocytes. More detailed insights into the MBP synthesis pathway are important for a better understanding of the myelination process and may foster the development of remyelination therapies for demyelinating diseases.

  13. Induction of human spermine oxidase SMO(PAOh1) is regulated at the levels of new mRNA synthesis, mRNA stabilization and newly synthesized protein.

    Science.gov (United States)

    Wang, Yanlin; Hacker, Amy; Murray-Stewart, Tracy; Fleischer, Jennifer G; Woster, Patrick M; Casero, Robert A

    2005-03-15

    The oxidation of polyamines induced by antitumour polyamine analogues has been associated with tumour response to specific agents. The human spermine oxidase, SMO(PAOh1), is one enzyme that may play a direct role in the cellular response to the antitumour polyamine analogues. In the present study, the induction of SMO(PAOh1) enzyme activity by CPENSpm [N1-ethyl-N11-(cyclopropyl)methyl-4,8,diazaundecane] is demonstrated to be a result of newly synthesized mRNA and protein. Inhibition of new RNA synthesis by actinomycin D inhibits both the appearance of SMO(PAOh1) mRNA and enzyme activity. Similarly, inhibition of newly synthesized protein with cycloheximide prevents analogue-induced enzyme activity. Half-life determinations indicate that stabilization of SMO(PAOh1) protein does not play a significant role in analogue-induced activity. However, half-life experiments using actinomycin D indicate that CPENSpm treatment not only increases mRNA expression, but also leads to a significant increase in mRNA half-life (17.1 and 8.8 h for CPENSpm-treated cells and control respectively). Using reporter constructs encompassing the SMO(PAOh1) promoter region, a 30-90% increase in transcription is observed after exposure to CPENSpm. The present results are consistent with the hypothesis that analogue-induced expression of SMO(PAOh1) is a result of increased transcription and stabilization of SMO(PAOh1) mRNA, leading to increased protein production and enzyme activity. These data indicate that the major level of control of SMO(PAOh1) expression in response to polyamine analogues exposure is at the level of mRNA.

  14. Equalizing the information amounts of protein and mRNA by information theory.

    Science.gov (United States)

    Adiguzel, Y

    2017-09-01

    Based on the Shannon's information communication theory, information amount of the entire length of a polymeric macromolecule can be calculated in bits through adding the entropies of each building block. Proteins, DNA and RNA are such macromolecules. When only the building blocks' variation is considered as the source of entropy, there is seemingly lower information in case of the protein if this approach is applied directly on a protein of specific size and the coding sequence size of the mRNA corresponding to the particular length of the protein. This decrease in the information amount seems contradictory but this apparent conflict is resolved by considering the conformational variations in proteins as a new variable in the calculation and balancing the approximated entropy of the coding part of the mRNA and the protein. Probabilities can change therefore we also assigned hypothetical probabilities to the conformational states, which represent the uneven distribution as the time spent in one conformation, providing the probability of the presence in either or one of the possible conformations. Results that are obtained by using hypothetical probabilities are in line with the experimental values of variations in the conformational-state of protein populations. This equalization approach has further biological relevance that it compensates for the degeneracy in the codon usage during protein translation and it leads to the conclusion that the alphabet size for the protein is rather optimal for the proper protein functioning within the thermodynamic milieu of the cell. The findings were also discussed in relation to the codon bias and have implications in relation to the codon evolution concept. Eventually, this work brings the fields of protein structural studies and molecular protein translation processes together with a novel approach. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Dysregulation of human beta-defensin-2 protein in inflammatory bowel disease.

    Directory of Open Access Journals (Sweden)

    Marian C Aldhous

    Full Text Available BACKGROUND: Human beta-defensin-2 (HBD2 is an antimicrobial peptide implicated in the pathogenesis of inflammatory bowel disease (IBD. Low copy number and concomitant low mRNA expression of the HBD2 gene have been implicated in susceptibility to colonic Crohn's Disease (CD. We investigated the colonic distribution of HBD2 mRNA expression, and the contributions of genetic and environmental factors on HBD2 protein production. METHODOLOGY/PRINCIPAL FINDINGS: We examined HBD2 mRNA expression at three colonic locations by microarray analysis of biopsies from 151 patients (53 CD, 67 ulcerative colitis [UC], 31 controls. We investigated environmental and genetic influences on HBD2 protein production using ex vivo cultured sigmoid colon biopsies from 69 patients (22 CD, 26 UC, 21 controls stimulated with lipopolysaccharide (LPS and/or nicotine for 24 hours. HBD2 and cytokines were measured in culture supernatants. Using DNA samples from these patients, regions in the HBD2 gene promoter were sequenced for NF-kappaB binding-sites and HBD2 gene copy number was determined. HBD2 mRNA expression was highest in inflamed (vs. uninflamed p = 0.0122 ascending colon in CD and in inflamed (vs. uninflamed p<0.0001 sigmoid colon in UC. HBD2 protein production was increased in inflamed UC biopsies (p = 0.0078. There was no difference in HBD2 protein production from unstimulated biopsies of CD, UC and controls. LPS-induced HBD2 production was significantly increased in CD (p = 0.0375 but not UC (p = 0.2017; this LPS-induced response was augmented by nicotine in UC (p = 0.0308 but not CD (p = 0.6872. Nicotine alone did not affect HBD2 production. HBD2 production correlated with IL8 production in UC (p<0.001 and with IL10 in CD (p<0.05. Variations in the HBD2 promoter and HBD2 gene copy number did not affect HBD2 production. SIGNIFICANCE/CONCLUSIONS: Colonic HBD2 was dysregulated at mRNA and protein level in IBD. Inflammatory status and stimulus but not germline

  16. beta(2)microglobulin mRNA expression levels are prognostic for lymph node metastasis in colorectal cancer patients.

    Science.gov (United States)

    Shrout, J; Yousefzadeh, M; Dodd, A; Kirven, K; Blum, C; Graham, A; Benjamin, K; Hoda, R; Krishna, M; Romano, M; Wallace, M; Garrett-Mayer, E; Mitas, M

    2008-06-17

    Colorectal cancer (CRC) is the fourth most common non-cutaneous malignancy in the United States and the second most frequent cause of cancer-related death. One of the most important determinants of CRC survival is lymph node metastasis. To determine whether molecular markers might be prognostic for lymph node metastases, we measured by quantitative real-time RT-PCR the expression levels of 15 cancer-associated genes in formalin-fixed paraffin-embedded primary tissues derived from stage I-IV CRC patients with (n=20) and without (n=18) nodal metastases. Using the mean of the 15 genes as an internal reference control, we observed that low expression of beta(2)microglobulin (B2M) was a strong prognostic indicator of lymph node metastases (area under the curve (AUC)=0.85; 95% confidence interval (CI)=0.69-0.94). We also observed that the expression ratio of B2M/Spint2 had the highest prognostic accuracy (AUC=0.87; 95% CI=0.71-0.96) of all potential two-gene combinations. Expression values of Spint2 correlated with the mean of the entire gene set at an R(2) value of 0.97, providing evidence that Spint2 serves not as an independent prognostic gene, but rather as a reliable reference control gene. These studies are the first to demonstrate a prognostic role of B2M at the mRNA level and suggest that low B2M expression levels might be useful for identifying patients with lymph node metastasis and/or poor survival.

  17. Proteins from rat liver cytosol which stimulate mRNA transport. Purification and interactions with the nuclear envelope mRNA translocation system.

    Science.gov (United States)

    Schröder, H C; Rottmann, M; Bachmann, M; Müller, W E; McDonald, A R; Agutter, P S

    1986-08-15

    Two polysome-associated proteins with particular affinities for poly(A) have been purified from rat liver. These proteins stimulate the efflux of mRNA from isolated nuclei in conditions under which such efflux closely stimulates mRNA transport in vivo, and they are therefore considered as mRNA-transport-stimulatory proteins. Their interaction with the mRNA-translocation system in isolated nuclear envelopes has been studied. The results are generally consistent with the most recently proposed kinetic model of mRNA translocation. One protein, P58, has not been described previously. It inhibits the protein kinase that down-regulates the NTPase, it enhances the NTPase activity in both the presence and the absence of poly(A) and it seems to increase poly(A) binding in unphosphorylated, but not in phosphorylated, envelopes. The other protein, P31, which probably corresponds to the 35,000-Mr factor described by Webb and his colleagues, enhances the binding of poly(A) to the mRNA-binding site in the envelope, thus stimulating the phosphoprotein phosphatase and, in consequence, the NTPase. The possible physiological significance of these two proteins is discussed.

  18. Vaccinia virus recombinants expressing an 11-kilodalton beta-galactosidase fusion protein incorporate active beta-galactosidase in virus particles.

    Science.gov (United States)

    Huang, C; Samsonoff, W A; Grzelecki, A

    1988-10-01

    Recombinant plasmids in which vaccinia virus transcriptional regulatory sequences were fused to the Escherichia coli lacZ gene were constructed for insertion of the lacZ gene into the vaccinia virus genome. beta-Galactosidase (beta-gal) was found in some purified recombinant vaccinia virions. By enzyme activity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and microscopic techniques, the evidence suggested that beta-gal accounted for 5% of the total protein in the virion. These recombinant viruses were constructed so that a portion of the coding sequences of a late vaccinia virus structural polypeptide was fused to the amino terminus of beta-gal to produce the fusion protein. Removal of the coding sequences resulted in the complete loss of beta-gal activity. This demonstrated that a vaccinia virus DNA segment from a late structural gene is responsible for the incorporation of beta-gal into the virion.

  19. Effect of physical training on glucose transporter protein and mRNA levels in rat adipocytes

    DEFF Research Database (Denmark)

    Stallknecht, B; Andersen, P H; Vinten, J

    1993-01-01

    Physical training increases insulin-stimulated glucose transport and the number of glucose transporters in adipocytes measured by cytochalasin B binding. In the present study we used immunoblotting to measure the abundance of two glucose transporters (GLUT-4, GLUT-1) in white adipocytes from....../or intrinsic activity). GLUT-1 protein and mRNA levels/adipocyte volume did not change with age or training....

  20. INCA, a novel human caspase recruitment domain protein that inhibits interleukin-1beta generation.

    Science.gov (United States)

    Lamkanfi, Mohamed; Denecker, Geertrui; Kalai, Michael; D'hondt, Kathleen; Meeus, Ann; Declercq, Wim; Saelens, Xavier; Vandenabeele, Peter

    2004-12-10

    Using in silico methods for screening the human genome for new caspase recruitment domain (CARD) proteins, we have identified INCA (Inhibitory CARD) as a protein that shares 81% identity with the prodomain of caspase-1. The INCA gene is located on chromosome 11q22 between the genes of COP/Pseudo-ICE and ICEBERG, two other CARD proteins that arose from caspase-1 gene duplications. We show that INCA mRNA is expressed in many tissues. INCA is specifically upregulated by interferon-gamma in the monocytic cell lines THP-1 and U937. INCA physically interacts with procaspase-1 and blocks the release of mature IL-1beta from LPS-stimulated macrophages. Unlike COP/Pseudo-ICE and procaspase-1, INCA does not interact with RIP2 and does not induce NF-kappaB activation. Our data show that INCA is a novel intracellular regulator of procaspase-1 activation, involved in the regulation of pro-IL-1beta processing and its release during inflammation.

  1. Expression of mRNA and protein-protein interaction of the antiviral endoribonuclease RNase L in mouse spleen.

    Science.gov (United States)

    Gupta, Ankush; Rath, Pramod C

    2014-08-01

    The interferon-inducible, 2',5'-oligoadenylate (2-5A)-dependent endoribonuclease, RNase L is a unique antiviral RNA-degrading enzyme involved in RNA-metabolism, translational regulation, stress-response besides its anticancer/tumor-suppressor and antibacterial functions. RNase L represents complex cellular RNA-regulations in mammalian cells but diverse functions of RNase L are not completely explained by its 2-5A-regulated endoribonuclease activity. We hypothesized that RNase L has housekeeping function(s) through interaction with cellular proteins. We investigated RNase L mRNA expression in mouse tissues by RT-PCR and its protein-protein interaction in spleen by GST-pulldown and immunoprecipitation assays followed by proteomic analysis. RNase L mRNA is constitutively and differentially expressed in nine different mouse tissues, its level is maximum in immunological tissues (spleen, thymus and lungs), moderate in reproductive tissues (testis and prostate) and low in metabolic tissues (kidney, brain, liver and heart). Cellular proteins from mouse spleen [fibronectin precursor, β-actin, troponin I, myosin heavy chain 9 (non-muscle), growth-arrest specific protein 11, clathrin light chain B, a putative uncharacterized protein (Ricken cDNA 8030451F13) isoform (CRA_d) and alanyl tRNA synthetase] were identified as cellular RNase L-interacting proteins. Thus our results suggest for more general cellular functions of RNase L through protein-protein interactions in the spleen for immune response in mammals.

  2. Collagen V-induced nasal tolerance downregulates pulmonary collagen mRNA gene and TGF-beta expression in experimental systemic sclerosis

    Directory of Open Access Journals (Sweden)

    Parra Edwin R

    2010-01-01

    Full Text Available Abstract Background The purpose of this study was to evaluate collagen deposition, mRNA collagen synthesis and TGF-beta expression in the lung tissue in an experimental model of scleroderma after collagen V-induced nasal tolerance. Methods Female New Zealand rabbits (N = 12 were immunized with 1 mg/ml of collagen V in Freund's adjuvant (IM. After 150 days, six immunized animals were tolerated by nasal administration of collagen V (25 μg/day (IM-TOL daily for 60 days. The collagen content was determined by morphometry, and mRNA expressions of types I, III and V collagen were determined by Real-time PCR. The TGF-beta expression was evaluated by immunostaining and quantified by point counting methods. To statistic analysis ANOVA with Bonferroni test were employed for multiple comparison when appropriate and the level of significance was determined to be p Results IM-TOL, when compared to IM, showed significant reduction in total collagen content around the vessels (0.371 ± 0.118 vs. 0.874 ± 0.282, p p p = 0.026. The lung tissue of IM-TOL, when compared to IM, showed decreased immunostaining of types I, III and V collagen, reduced mRNA expression of types I (0.10 ± 0.07 vs. 1.0 ± 0.528, p = 0.002 and V (1.12 ± 0.42 vs. 4.74 ± 2.25, p = 0.009 collagen, in addition to decreased TGF-beta expression (p Conclusions Collagen V-induced nasal tolerance in the experimental model of SSc regulated the pulmonary remodeling process, inhibiting collagen deposition and collagen I and V mRNA synthesis. Additionally, it decreased TGF-beta expression, suggesting a promising therapeutic option for scleroderma treatment.

  3. Tumor necrosis factor-alpha upregulates 11beta-hydroxysteroid dehydrogenase type 1 expression by CCAAT/enhancer binding protein-beta in HepG2 cells.

    Science.gov (United States)

    Ignatova, Irena D; Kostadinova, Radina M; Goldring, Christopher E; Nawrocki, Andrea R; Frey, Felix J; Frey, Brigitte M

    2009-02-01

    The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the conversion of inactive to active glucocorticoids. 11beta-HSD1 plays a crucial role in the pathogenesis of obesity and controls glucocorticoid actions in inflammation. Several studies have demonstrated that TNF-alpha increases 11beta-HSD1 mRNA and activity in various cell models. Here, we demonstrate that mRNA and activity of 11beta-HSD1 is increased in liver tissue from transgenic mice overexpressing TNF-alpha, indicating that this effect also occurs in vivo. To dissect the molecular mechanism of this increase, we investigated basal and TNF-alpha-induced transcription of the 11beta-HSD1 gene (HSD11B1) in HepG2 cells. We found that TNF-alpha acts via p38 MAPK pathway. Transient transfections with variable lengths of human HSD11B1 promoter revealed highest activity with or without TNF-alpha in the proximal promoter region (-180 to +74). Cotransfection with human CCAAT/enhancer binding protein-alpha (C/EBPalpha) and C/EBPbeta-LAP expression vectors activated the HSD11B1 promoter with the strongest effect within the same region. Gel shift and RNA interference assays revealed the involvement of mainly C/EBPalpha, but also C/EBPbeta, in basal and only of C/EBPbeta in the TNF-alpha-induced HSD11B1 expression. Chromatin immunoprecipitation assay confirmed in vivo the increased abundance of C/EBPbeta on the proximal HSD11B1 promoter upon TNF-alpha treatment. In conclusion, C/EBPalpha and C/EBPbeta control basal transcription, and TNF-alpha upregulates 11beta-HSD1, most likely by p38 MAPK-mediated increased binding of C/EBPbeta to the human HSD11B1 promoter. To our knowledge, this is the first study showing involvement of p38 MAPK in the TNF-alpha-mediated 11beta-HSD1 regulation, and that TNF-alpha stimulates enzyme activity in vivo.

  4. Oligodendrocyte transcription factor 1 mRNA and protein expression in organotypic rat brain slices

    Institute of Scientific and Technical Information of China (English)

    Hong Cui; Lijun Yang; Dezhuang Huang; Wandong Zhang; Weijuan Han; Yanqing Yao; Wenxing Jiang

    2010-01-01

    Numerous studies have confirmed that oligodendrocyte transcription factor 1 (Olig-1) is vital for myelin repair. However, the effects of hypoxia and ischemia on Olig-1 expression remain unknown.In this study, Olig-1 mRNA and protein expressions were analyzed by in situ hybridization and immunohistochemistry, to determine the expression profile of Olig-1 in rat brain slices exposed to hypoxia and ischemia. Brains were obtained from 2-day-old Sprague-Dawley rats, and sections were randomly assigned to control and hypoxia/ischemia groups. Hematoxylin-eosin staining revealed karyorrhexis and karyopyknosis in cells from the hypoxia/ischemia group. Under electron microscopy, mitochondria swelling and neuropil edema were observed in the hypoxia/ischemia group. Olig-1 mRNA and protein expressions were increased at 1 day after hypoxia and ischemia treatment. These results suggest that in situ hybridization and immunohistochemistry could be used simultaneously to detect mRNA and protein expression in brain slices.

  5. In vivo cross-linking followed by polyA enrichment to identify yeast mRNA binding proteins.

    Science.gov (United States)

    Mitchell, Sarah F; Parker, Roy

    2015-01-01

    mRNA binding proteins regulate gene expression by controlling the processing, localization, decay, and translation of messenger RNAs (mRNAs). To fully understand this process, it is necessary to identify the complete set of mRNA binding proteins. This work describes a method for the systematic identification of yeast mRNA binding proteins. This method applies in vivo UV cross-linking, affinity pull-down of polyA(+) mRNAs, and analysis by mass spectrometry to identify proteins that directly bind to mRNAs.

  6. Eosinophil cationic protein mRNA expression in children with bronchial asthma.

    Science.gov (United States)

    Yu, H Y; Li, X Y; Cai, Z F; Li, L; Shi, X Z; Song, H X; Liu, X J

    2015-11-13

    Studies have shown that eosinophils are closely related to pathogenesis of bronchial asthma. Eosinophils release eosinophil cationic protein (ECP), which plays an important role in infection and allergic reactions. Serum ECP mRNA expression in children with bronchial asthma has not been adequately investigated. We analyzed serum ECP mRNA expression in 63 children with bronchial asthma and 21 healthy children by using reverse-transcriptase polymerase chain reaction to understand the role of ECP in children with bronchial asthma. The children with bronchial asthma were segregated into acute-phase and stable-phase groups, based on the severity of the illness. Serum ECP mRNA expression in children with bronchial asthma (0.375 ± 0.04) was significantly higher than that in healthy controls (0.20 ± 0.02; P children in the acute-phase group showed higher ECP mRNA expression level (0.44 ± 0.06) than those in the stable-phase (0.31 ± 0.03) and healthy control groups (0.20 ± 0.02; P children with bronchial asthma.

  7. mRNA Expression of Ovine Angiopoietin-like Protein 4 Gene in Adipose Tissues.

    Science.gov (United States)

    Zhang, Jing; Jing, Jiong-Jie; Jia, Xia-Li; Qiao, Li-Ying; Liu, Jian-Hua; Liang, Chen; Liu, Wen-Zhong

    2016-05-01

    Angiopoietin-like protein 4 (ANGPTL4) is involved in a variety of functions, including lipoprotein metabolism and angiogenesis. To reveal the role of ANGPTL4 in fat metabolism of sheep, ovine ANGPTL4 mRNA expression was analyzed in seven adipose tissues from two breeds with distinct tail types. Forty-eight animals with the gender ratio of 1:1 for both Guangling Large Tailed (GLT) and Small Tailed Han (STH) sheep were slaughtered at 2, 4, 6, 8, 10, and 12 months of age, respectively. Adipose tissues were collected from greater and lesser omental, subcutaneous, retroperitoneal, perirenal, mesenteric, and tail fats. Ontogenetic mRNA expression of ANGPTL4 in these adipose tissues from GTL and STH was studied by quantitative real time polymerase chain reaction. The results showed that ANGPTL4 mRNA expressed in all adipose tissues studied with the highest in subcutaneous and the lowest in mesenteric fat depots. Months of age, tissue and breed are the main factors that significantly influence the mRNA expression. These results provide new insights into ovine ANGPTL4 gene expression and clues for its function mechanism.

  8. Effect of prolonged exposure to sublethal concentrations of DDT and DDE on protein expression in human pancreatic beta cells.

    Science.gov (United States)

    Pavlikova, Nela; Smetana, Pavel; Halada, Petr; Kovar, Jan

    2015-10-01

    Pollution of the environment represents one of less explored potential reasons for the worldwide epidemic of type 2 diabetes. One of the most prevalent organochlorine pollutants remains the pesticide DDT and its degradation product DDE. Despite some epidemiologic correlations between levels of DDT and DDE in human organism and the prevalence of diabetes, there is almost no information about the exact targets of these compounds inside pancreatic beta cells. To detect functional areas of pancreatic beta cells that could be affected by exposure to DDT and DDE, we analyzed changes in protein expression in the NES2Y human pancreatic beta cell line exposed to three sublethal concentrations (0.1 μM, 1 μM, 10 μM) of DDT and DDE for 1 month. Protein separation and identification was achieved using high-resolution 2D-electrophoresis, computer analysis and mass spectrometry. With these techniques, four proteins were found downregulated after exposure to 10 μM DDT: three cytoskeletal proteins (cytokeratin 8, cytokeratin 18 and actin) and one protein involved in glycolysis (alpha-enolase). Two proteins were downregulated after exposure to 10 μM DDE: cytokeratin 18 and heterogenous nuclear ribonucleoprotein H1 (HNRH1). These changes correlate with previously described effects of other stress conditions (e.g. exposure to palmitate, hyperglycemia, imidazoline derivative, and cytokines) on protein expression in pancreatic beta cells. We conclude that cytoskeletal proteins and their processing, glucose metabolism, and mRNA processing may represent targets affected by exposure to conditions hostile to pancreatic beta cells, including exposure to DDT and DDE.

  9. Simultaneous detection of mRNA and protein stem cell markers in live cells

    Directory of Open Access Journals (Sweden)

    Bao Gang

    2009-04-01

    Full Text Available Abstract Background Biological studies and medical application of stem cells often require the isolation of stem cells from a mixed cell population, including the detection of cancer stem cells in tumor tissue, and isolation of induced pluripotent stem cells after eliciting the expression of specific genes in adult cells. Here we report the detection of Oct-4 mRNA and SSEA-1 protein in live carcinoma stem cells using respectively molecular beacon and dye-labeled antibody, aiming to establish a new method for stem cells detection and isolation. Results Quantification of Oct-4 mRNA and protein in P19 mouse carcinoma stem cells using respectively RT-PCR and immunocytochemistry confirmed that their levels drastically decreased after differentiation. To visualize Oct-4 mRNA in live stem cells, molecular beacons were designed, synthesized and validated, and the detection specificity was confirmed using control studies. We found that the fluorescence signal from Oct-4-targeting molecular beacons provides a clear discrimination between undifferentiated and retinoic acid-induced differentiated cells. Using deconvolution fluorescence microscopy, Oct-4 mRNAs were found to reside on one side of the cytosol. We demonstrated that, using a combination of Oct-4 mRNA-targeting molecular beacon with SSEA-1 antibody in flow cytometric analysis, undifferentiated stem cells can be clearly distinguished from differentiated cells. We revealed that Oct-4 targeting molecular beacons do not seem to affect stem cell biology. Conclusion Molecular beacons have the potential to provide a powerful tool for highly specific detection and isolation of stem cells, including cancer stem cells and induced pluripotent stem (iPS cells without disturbing cell physiology. It is advantageous to perform simultaneous detection of intracellular (mRNA and cell-surface (protein stem cell markers in flow cytometric analysis, which may lead to high detection sensitivity and efficiency.

  10. Characterizing exogenous mRNA delivery, trafficking, cytoplasmic release and RNA-protein correlations at the level of single cells.

    Science.gov (United States)

    Kirschman, Jonathan L; Bhosle, Sushma; Vanover, Daryll; Blanchard, Emmeline L; Loomis, Kristin H; Zurla, Chiara; Murray, Kathryn; Lam, Blaine C; Santangelo, Philip J

    2017-07-07

    The use of synthetic messenger ribonucleic acid (mRNA) to express specific proteins is a highly promising therapeutic and vaccine approach that avoids many safety issues associated with viral or DNA-based systems. However, in order to optimize mRNA designs and delivery, technology advancements are required to study fundamental mechanisms of mRNA uptake and localization at the single-cell and tissue level. Here, we present a single RNA sensitive fluorescent labeling method which allows us to label and visualize synthetic mRNA without significantly affecting function. This approach enabled single cell characterization of mRNA uptake and release kinetics from endocytic compartments, the measurement of mRNA/protein correlations, and motivated the investigation of mRNA induced cellular stress, all important mechanisms influencing protein production. In addition, we demonstrated this approach can facilitate near-infrared imaging of mRNA localization in vivo and in ex-vivo tissue sections, which will facilitate mRNA trafficking studies in pre-clinical models. Overall, we demonstrate the ability to study fundamental mechanisms necessary to optimize delivery and therapeutic strategies, in order to design the next generation of novel mRNA therapeutics and vaccines. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. RNA mutagenesis yields highly diverse mRNA libraries for in vitro protein evolution

    Directory of Open Access Journals (Sweden)

    Dobric Nada

    2007-04-01

    Full Text Available Abstract Background In protein drug development, in vitro molecular optimization or protein maturation can be used to modify protein properties. One basic approach to protein maturation is the introduction of random DNA mutations into the target gene sequence to produce a library of variants that can be screened for the preferred protein properties. Unfortunately, the capability of this approach has been restricted by deficiencies in the methods currently available for random DNA mutagenesis and library generation. Current DNA based methodologies generally suffer from nucleotide substitution bias that preferentially mutate particular base pairs or show significant bias with respect to transitions or transversions. In this report, we describe a novel RNA-based random mutagenesis strategy that utilizes Qβ replicase to manufacture complex mRNA libraries with a mutational spectrum that is close to the ideal. Results We show that Qβ replicase generates all possible base substitutions with an equivalent preference for mutating A/T or G/C bases and with no significant bias for transitions over transversions. To demonstrate the high diversity that can be sampled from a Qβ replicase-generated mRNA library, the approach was used to evolve the binding affinity of a single domain VNAR shark antibody fragment (12Y-2 against malarial apical membrane antigen-1 (AMA-1 via ribosome display. The binding constant (KD of 12Y-2 was increased by 22-fold following two consecutive but discrete rounds of mutagenesis and selection. The mutagenesis method was also used to alter the substrate specificity of β-lactamase which does not significantly hydrolyse the antibiotic cefotaxime. Two cycles of RNA mutagenesis and selection on increasing concentrations of cefotaxime resulted in mutants with a minimum 10,000-fold increase in resistance, an outcome achieved faster and with fewer overall mutations than in comparable studies using other mutagenesis strategies

  12. Effect of running training on uncoupling protein mRNA expression in rat brown adipose tissue

    Science.gov (United States)

    Yamashita, Hitoshi; Yamamoto, Mikio; Sato, Yuzo; Izawa, Tetsuya; Komabayashi, Takao; Saito, Daizo; Ohno, Hideki

    1993-03-01

    The effect was investigated of endurance training on the expression of uncoupling protein (UCP) mRNA in brown adipose tissue (BAT) of rats. The exercised rats were trained on a rodent treadmill for 5 days per week and a total of 9 weeks. After the training programme, a marked decrease in BAT mass was found in terms of weight or weight per unit body weight; there was a corresponding decrease in DNA content and a downward trend in RNA and glycogen levels. The UCP mRNA was present at a markedly decreased level in BAT of trained animals. In consideration of the reduced levels of mRNAs for hormone-sensitive lipase and acylCoA synthetase, the brown adipose tissue investigated appeared to be in a relatively atrophied and thermogenically quiescent state.

  13. Translational pauses during the synthesis of proteins and mRNA structure.

    Science.gov (United States)

    Zama, M

    1997-01-01

    Translational pauses are observed during a spider fibroin synthesis (1,2). The spider major ampullate (dragline) silk of the spider Nephila clavipes is composed of multiple proteins. The amino acid sequences of the partial cDNA clones for the two major dragline silk fibroin components (Spidroin 1 and 2) exhibit repetitive motifs (3,4). Our detailed inspection of the nucleotide sequences of the repetitive motifs revealed highly selective site-specific codon usage patterns within a motif, suggesting that the secondary structure of the spider fibroin mRNA is optimized by the nucleotide sequence of the fibroin gene. The results, combined with our preceding results on silk fibroin from Bombyx mori (5) suggest that translational pauses of spider silk are interpreted in terms of the mRNA secondary structure.

  14. Expression of connexin 43 mRNA and protein in developing follicles of prepubertal porcine ovaries

    Science.gov (United States)

    Melton, C.M.; Zaunbrecher, G.M.; Yoshizaki, G.; Patio, R.; Whisnant, S.; Rendon, A.; Lee, V.H.

    2001-01-01

    A major form of cell-cell communication is mediated by gap junctions, aggregations of intercellular channels composed of connexins (Cxs), which are responsible for exchange of low molecular weight (hybridization revealed that Cx43 mRNA was detectable in granulosa cells of primary follicles but undetectable in dormant primordial follicles. The intensity of the signal increased with follicular growth and was greatest in the large antral follicles. Immunohistochemical evaluation indicated that Cx43 protein expression correlated with the presence of Cx43 mRNA. These results indicate that substantial amounts of Cx43 are first expressed in granulosa cells following activation of follicular development and that this expression increases throughout follicular growth and maturation. These findings suggest an association between the enhancement of intercellular gap-junctional communication and onset of follicular growth. ?? 2001 Elsevier Science Inc. All rights reserved.

  15. Development of K562 cell clones expressing beta-globin mRNA carrying the beta039 thalassaemia mutation for the screening of correctors of stop-codon mutations.

    Science.gov (United States)

    Salvatori, Francesca; Cantale, Vera; Breveglieri, Giulia; Zuccato, Cristina; Finotti, Alessia; Bianchi, Nicoletta; Borgatti, Monica; Feriotto, Giordana; Destro, Federica; Canella, Alessandro; Breda, Laura; Rivella, Stefano; Gambari, Roberto

    2009-07-09

    Nonsense mutations, giving rise to UAA, UGA and UAG stop codons within the coding region of mRNAs, promote premature translational termination and are the leading cause of approx. 30% of inherited diseases, including cystic fibrosis, Duchenne muscular dystrophy and thalassaemia. For instance, in beta(0)39-thalassaemia the CAG (glutamine) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well-described NMD (nonsense-mediated mRNA decay). In order to develop an approach facilitating translation and, therefore, protection from NMD, aminoglycoside antibiotics have been tested on mRNAs carrying premature stop codons. These drugs decrease the accuracy in the codon-anticodon base-pairing, inducing a ribosomal read-through of the premature termination codons. Interestingly, recent papers have described drugs designed and produced for suppressing premature translational termination, inducing a ribosomal read-through of premature but not normal termination codons. These findings have introduced new hopes for the development of a pharmacological approach to the therapy of beta(0)39-thalassaemia. In this context, we started the development of a cellular model of the beta(0)39-thalassaemia mutation that could be used for the screening of a high number of aminoglycosides and analogous molecules. To this aim, we produced a lentiviral construct containing the beta(0)39-thalassaemia globin gene under a minimal LCR (locus control region) control and used this construct for the transduction of K562 cells, subsequently subcloned, with the purpose to obtain several K562 clones with different integration copies of the construct. These clones were then treated with Geneticin (also known as G418) and other aminoglycosides and the production of beta-globin was analysed by FACS analysis. The results obtained suggest that this experimental system is suitable for the characterization of correction of the beta(0

  16. Sm proteins specify germ cell fate by facilitating oskar mRNA localization

    Science.gov (United States)

    Gonsalvez, Graydon B.; Rajendra, T. K.; Wen, Ying; Praveen, Kavita; Matera, A. Gregory

    2010-01-01

    Sm and Sm-like proteins are RNA-binding factors found in all three domains of life. Eukaryotic Sm proteins play essential roles in pre-mRNA splicing, forming the cores of spliceosomal small nuclear ribonucleoproteins (snRNPs). Recently, Sm proteins have been implicated in the specification of germ cells. However, a mechanistic understanding of their involvement in germline specification is lacking and a germline-specific RNA target has not been identified. We demonstrate that Drosophila SmB and SmD3 are specific components of the oskar messenger ribonucleoprotein (mRNP), proper localization of which is required for establishing germline fate and embryonic patterning. Importantly, oskar mRNA is delocalized in females harboring a hypomorphic mutation in SmD3, and embryos from mutant mothers are defective in germline specification. We conclude that Sm proteins function to establish the germline in Drosophila, at least in part by mediating oskar mRNA localization. PMID:20570937

  17. The ticking tail: daily oscillations in mRNA poly(A) tail length drive circadian cycles in protein synthesis.

    Science.gov (United States)

    Gotic, Ivana; Schibler, Ueli

    2012-12-15

    In this issue of Genes & Development, Kojima and colleagues (pp. 2724-2736) examined the impact of mRNA poly(A) tail length on circadian gene expression. Their study demonstrates how dynamic changes in transcript poly(A) tail length can lead to rhythmic protein expression, irrespective of whether mRNA accumulation is circadian or constitutive.

  18. Differential expression of melanopsin mRNA and protein in Brown Norwegian rats.

    Science.gov (United States)

    Hannibal, Jens; Georg, Birgitte; Fahrenkrug, Jan

    2013-01-01

    Melanopsin is expressed in a subpopulation of retinal ganglion cells rendering these cells intrinsically photosensitive (ipRGCs). The ipRGCs are the primary RGCs mediating light entrainment of the circadian clock and control of the pupillary light reflex, light regulated melatonin secretion and negative masking behaviour. Previous studies have demonstrated that melanopsin expression in albino rats is regulated by light and darkness. The present study was undertaken to study the influence of light and darkness during the circadian day and after extended periods of constant light and darkness on melanopsin expression in the pigmented retina of the Brown Norwegian rat (Rattus norvegicus). The diurnal and circadian expressions were examined in retinal extracts from rats euthanized every 4 h during a 24 h light/dark (LD) and a 24 h dark cycle (DD) using quantitative real-time PCR and Western blotting. To study whether light regulates melanopsin expression, rats were sacrificed after being placed in either constant light (LL) or darkness for 3 or 21 d. Flat mount retinas from animals kept during either LL or DD were also examined by immunohistochemistry. Melanopsin mRNA expression displayed a significant rhythmic change during the LD cycle with peak expression around dusk and nadir at dawn. Melanopsin protein also changed over the LD cycle with peak expression at the end of the night and nadir at dusk. Rhythmic expression of melanopsin mRNA but not melanopsin protein was found in constant darkness. After 3 or 21 d in either LL or DD melanopsin mRNA expression was unaltered. Melanopsin protein was at the same high level after 3 and 21 d in DD, whereas a significant decrease was found after prolonging the light period for 3 or 21 d. The change in melanopsin protein was primarily due to change in immunoreactivity in the dendritic processes. In conclusion we found that light and darkness are important for regulation of melanopsin protein expression whereas input from a

  19. Cloning and Characterization of an mRNA Encoding F1-ATPase Beta-Subunit Abundant in Epithelial Cells of Mantle and Gill of Pearl Oyster, Pinctadafucata

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    In oyster biomineralization, large amounts of calcium are absorbed from external media, transported to the mineralization site, and finally deposited via a matrix-mediated process. All these activities are very energy intensive; therefore, investigations of the energy metabolism pathways of different oyster tissues will facilitate understanding of oyster biomineralization physiology. A full-length cDNA encoding the F1-ATPase beta-subunit (the F1-β-subunit, a major calalytic subunit of F-ATPase) from the pearl oyster (Pinctads fucata) was cloned using the homology strategy with a pair of degenerated primers based on the conserved regions of other animals' F1-β-subunit genes. Sequencing and structural analyses showed that the obtained sequence shared high identity with other animals' F1-β-subunits, and had a unique phosphorylation site of PKC and CK Ⅱ on the external surface of the putative protein. Results from semi-quantitative reverse transcription-polymerase chain reaction and in situ hybridization demonstrated this oyster F1-β-subunit mRNA is abundant in the gill and mantle, and distributed widely in the periostracal groove, the outer folder,and the dorsal region of the mantle and in the gill epithelial cells. These tissues were the main regions that participate in biomineralization processes such as calcium uptake, transport, and matrix secretion. The results indicate that tissues involved in biomineralization have stronger energy metabolic processes and that F1-ATPase might play an important role in oyster biomineralization by providing energy transport.

  20. Discovery of Proteomic Code with mRNA Assisted Protein Folding

    Directory of Open Access Journals (Sweden)

    Jan C. Biro

    2008-12-01

    Full Text Available The 3x redundancy of the Genetic Code is usually explained as a necessity to increase the mutation-resistance of the genetic information. However recent bioinformatical observations indicate that the redundant Genetic Code contains more biological information than previously known and which is additional to the 64/20 definition of amino acids. It might define the physico-chemical and structural properties of amino acids, the codon boundaries, the amino acid co-locations (interactions in the coded proteins and the free folding energy of mRNAs. This additional information, which seems to be necessary to determine the 3D structure of coding nucleic acids as well as the coded proteins, is known as the Proteomic Code and mRNA Assisted Protein Folding.

  1. The Nuclear PolyA-Binding Protein Nab2p Is Essential for mRNA Production

    DEFF Research Database (Denmark)

    Schmid, Manfred; Olszewski, Pawel; Pelechano, Vicent

    2015-01-01

    Polyadenylation of mRNA is a key step in eukaryotic gene expression. However, despite the major impact of poly(A) tails on mRNA metabolism, the precise roles of poly(A)-binding proteins (PABPs) in nuclear mRNA biogenesis remain elusive. Here, we demonstrate that rapid nuclear depletion of the S....... cerevisiae PABP Nab2p leads to a global loss of cellular mRNA, but not of RNA lacking poly(A) tails. Disappearance of mRNA is a nuclear event, but not due to decreased transcription. Instead, the absence of Nab2p results in robust nuclear mRNA decay by the ribonucleolytic RNA exosome in a polyadenylation...

  2. Exercise training does not increase muscle FNDC5 protein or mRNA expression in pigs.

    Science.gov (United States)

    Fain, John N; Company, Joseph M; Booth, Frank W; Laughlin, M Harold; Padilla, Jaume; Jenkins, Nathan T; Bahouth, Suleiman W; Sacks, Harold S

    2013-10-01

    Exercise training elevates circulating irisin and induces the expression of the FNDC5 gene in skeletal muscles of mice. Our objective was to determine whether exercise training also increases FNDC5 protein or mRNA expression in the skeletal muscles of pigs as well as plasma irisin. Castrated male pigs of the Rapacz familial hypercholesterolemic (FHM) strain and normal (Yucatan miniature) pigs were sacrificed after 16-20 weeks of exercise training. Samples of cardiac muscle, deltoid and triceps brachii muscle, subcutaneous and epicardial fat were obtained and FNDC5 mRNA, along with that of 6 other genes, was measured in all tissues of FHM pigs by reverse transcription polymerase chain reaction. FNDC protein in deltoid and triceps brachii was determined by Western blotting in both FHM and normal pigs. Citrate synthase activity was measured in the muscle samples of all pigs as an index of exercise training. Irisin was measured by an ELISA assay. There was no statistically significant effect of exercise training on FNDC5 gene expression in epicardial or subcutaneous fat, deltoid muscle, triceps brachii muscle or heart muscle. Exercise-training elevated circulating levels of irisin in the FHM pigs and citrate synthase activity in deltoid and triceps brachii muscle. A similar increase in citrate synthase activity was seen in muscle extracts of exercise-trained normal pigs but there was no alteration in circulating irisin. Exercise training in pigs does not increase FNDC5 mRNA or protein in the deltoid or triceps brachii of FHM or normal pigs while increasing circulating irisin only in the FHM pigs. These data indicate that the response to exercise training in normal pigs is not comparable to that seen in mice. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. A FYVE zinc finger domain protein specifically links mRNA transport to endosome trafficking.

    Science.gov (United States)

    Pohlmann, Thomas; Baumann, Sebastian; Haag, Carl; Albrecht, Mario; Feldbrügge, Michael

    2015-05-18

    An emerging theme in cellular logistics is the close connection between mRNA and membrane trafficking. A prominent example is the microtubule-dependent transport of mRNAs and associated ribosomes on endosomes. This coordinated process is crucial for correct septin filamentation and efficient growth of polarised cells, such as fungal hyphae. Despite detailed knowledge on the key RNA-binding protein and the molecular motors involved, it is unclear how mRNAs are connected to membranes during transport. Here, we identify a novel factor containing a FYVE zinc finger domain for interaction with endosomal lipids and a new PAM2-like domain required for interaction with the MLLE domain of the key RNA-binding protein. Consistently, loss of this FYVE domain protein leads to specific defects in mRNA, ribosome, and septin transport without affecting general functions of endosomes or their movement. Hence, this is the first endosomal component specific for mRNP trafficking uncovering a new mechanism to couple mRNPs to endosomes.

  4. Early-life infection leads to altered BDNF and IL-1beta mRNA expression in rat hippocampus following learning in adulthood.

    Science.gov (United States)

    Bilbo, Staci D; Barrientos, Ruth M; Eads, Andrea S; Northcutt, Alexis; Watkins, Linda R; Rudy, Jerry W; Maier, Steven F

    2008-05-01

    Neonatal bacterial infection in rats leads to profound hippocampal-dependent memory impairments following a peripheral immune challenge in adulthood. Here, we determined whether neonatal infection plus an immune challenge in adult rats is associated with impaired induction of brain-derived neurotrophic factor (BDNF) within the hippocampus (CA1, CA3, and dentate gyrus) following fear conditioning. BDNF is well characterized for its critical role in learning and memory. Rats injected on postnatal day 4 with PBS (vehicle) or Escherichia coli received as adults either no conditioning or a single 2min trial of fear conditioning. Half of the rats in the conditioned group then received a peripheral injection of 25mug/kg lipopolysaccharide (LPS) and all were sacrificed 1 or 4h later. Basal (unconditioned) BDNF mRNA did not differ between groups. However, following conditioning, neonatal infection with E. coli led to decreased BDNF mRNA induction in all regions compared to PBS-treated rats. This decrease in E. coli-treated rats was accompanied by a large increase in IL-1beta mRNA in CA1. Taken together, these data indicate that early infection strongly influences the induction of IL-1beta and BDNF within distinct regions of the hippocampus, which likely contribute to observed memory impairments in adulthood.

  5. A Herpesvirus Protein Selectively Inhibits Cellular mRNA Nuclear Export.

    Science.gov (United States)

    Gong, Danyang; Kim, Yong Hoon; Xiao, Yuchen; Du, Yushen; Xie, Yafang; Lee, Kevin K; Feng, Jun; Farhat, Nisar; Zhao, Dawei; Shu, Sara; Dai, Xinghong; Chanda, Sumit K; Rana, Tariq M; Krogan, Nevan J; Sun, Ren; Wu, Ting-Ting

    2016-11-09

    Nuclear mRNA export is highly regulated to ensure accurate cellular gene expression. Viral inhibition of cellular mRNA export can enhance viral access to the cellular translation machinery and prevent anti-viral protein production but is generally thought to be nonselective. We report that ORF10 of Kaposi's sarcoma-associated herpesvirus (KSHV), a nuclear DNA virus, inhibits mRNA export in a transcript-selective manner to control cellular gene expression. Nuclear export inhibition by ORF10 requires an interaction with an RNA export factor, Rae1. Genome-wide analysis reveals a subset of cellular mRNAs whose nuclear export is blocked by ORF10 with the 3' UTRs of ORF10-targeted transcripts conferring sensitivity to export inhibition. The ORF10-Rae1 interaction is important for the virus to express viral genes and produce infectious virions. These results suggest that a nuclear DNA virus can selectively interfere with RNA export to restrict host gene expression for optimal replication. Published by Elsevier Inc.

  6. Power-frequency electromagnetic field promotes mRNA expressions of bone morphogenetic protein-2 and transforming growth factor-beta 1 in bone marrow mesenchymal stem cells%工频电磁场对小鼠骨髓间充质干细胞骨形态发生蛋白2和转化生长因子β1mRNA表达的刺激

    Institute of Scientific and Technical Information of China (English)

    陈继革; 吴华; 葛保健; 方真华

    2007-01-01

    BACKGROUND:Studies confirm that electromagnetic field (EMF) can promote the synthesis and secretion of many bone growth factors,and some growth factors can induce the osteoblastic directional differentiation of bone marrow mesenchymal stem cells (MSCs).OBJECTIVE: To investigate the effect of power-frequency EMF on mRNA expressions of bone morphogenetic protein-2 (BMP-2) and transforming growth factor-beta 1 (TGF-β1) in mouse bone marrow MSCs cultured in vitro.DESTGN: Single sample, block design, observation and controlled animal trial.SETTING: Department of Traumatic Surgery, Tongji Hospital Affiliated to Tongji Medical .College, Huazhong University of Science and Technology.MATERIALS: This trial was carried out in the laboratory of Department of Traumatic Surgery, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology during February to December 2005. ①Twenty Kunming mice of clean grade were selected for harvest of bone marrow MSCs. ②Magnetic field generator,which could generate EMF with 0 to 100 mT field strength and successive adjustable 50 Hz sinusoidal wave, was developed by Wuhan Naval University of Engineering. ③ Primer was all synthesized by Saibaisheng Bioengineering Co.,Ltd., Beijing.NETHODS: ① The involved mice were sacrificed by cervical dislocation. Bilateral femora and tibia were harvested. Bone marrow MSCs were isolated and cultured in vitro, and the second generation of cells were used for the trial. ②Different intensities of EMF stimulation tests: Negative control group, positive control group, EMF 0.4, 0.8 and 1.6 mT stimulation groups were set. Five bottles of cells of the second generation were chosen from each group for test. The cells in the negative control group and positive control group were not exposed to EMF. But medium containing osteogenic inductor(10-8 mol/L dexamethasone, 10 mmol/L β-sodium glycerophosphate and 50 mg/L Vitamin C included) was added in the positive control group

  7. Extended longevity lines of Drosophila melanogaster: abundance of yolk protein gene mRNA in fat body and ovary.

    Science.gov (United States)

    Carlson, K A; Harshman, L G

    1999-04-01

    Lines of Drosophila melanogaster selected for late-life female reproduction typically exhibit correlated responses of reduced early fecundity and increased longevity. This relationship suggests a tradeoff between reproductive effort and somatic maintenance, which in turn, underlies some evolutionary theories of senescence. The mechanistic basis of the apparent tradeoff between increased longevity and reduced early-age fecundity has remained obscure. The present manuscript addresses the issues of whether the reduced early-age fecundity in selected lines corresponds to reduced yolk-protein mRNA production, and whether long-lived flies exhibit somatic maintenance in terms of relatively reduced yolk-protein mRNA production in the fat body. Yolk protein is one of the most abundant proteins used for female reproduction. By comparing a set of lines selected for late life reproduction with the corresponding control lines, we show that that yolk-protein gene mRNA relative abundance during the first four days posteclosion did not correspond to reduced early-life fecundity in the selected lines. In D. melanogaster, yolk protein is produced in the fat body and ovarian follicle cells. On the fourth day posteclosion, relatively more yolk-protein gene mRNA was present in the fat body. On day 1 posteclosion, supplemental yeast did not alter relative yolk-protein gene mRNA abundance. However, on day 4 posteclosion, supplemental yeast stimulated yolk-protein gene mRNA production in the fat body, which suggests an underlying mechanism for the nutrition-based phenotypic plasticity of fecundity previously documented in these lines. On medium without supplemental yeast, the relatively low abundance of fat body yolk-protein gene mRNA in the selected lines on day 4 posteclosion corresponds to a prediction derived from the disposable soma theory.

  8. Phosphorylation of the mRNA cap binding protein and eIF-4A by different protein kinases

    Energy Technology Data Exchange (ETDEWEB)

    Hagedorn, C.H.

    1987-05-01

    These studies were done to determine the identity of a protein kinase that phosphorylates the mRNA cap binding protein (CBP). Two chromatographic steps (dye and ligand and ion exchange HPLC) produced a 500x purification of an enzyme activity in rabbit reticulocytes that phosphorylated CBP at serine residues. Isoelectric focusing analysis of kinase treated CBP demonstrated 5 isoelectric species of which the 2 most anodic species were phosphorylated (contained /sup 32/P). This kinase activity phosphorylated CBP when it was isolated or in the eIF-4F complex. Purified protein kinase C, cAMP or cGMP dependent protein kinase, casein kinase I or II, myosin light chain kinase or insulin receptor kinase did not significantly phosphorylate isolated CBP or CBP in the eIF-4F complex. However, cAMP and cGMP dependent protein kinases and casein kinase II phosphorylated eIF-4A but did not phosphorylate the 46 kDa component of eIF-4F. cAMP dependent protein kinase phosphorylated a approx. 220 kDa protein doublet in eIF-4F preparations. These studies indicate that CBP kinase activity probably represents a previously unidentified protein kinase. In addition, eIF-4A appears to be phosphorylated by several protein kinases whereas the 46 kDa component of the eIF-4F complex was not.

  9. Gastric acid induces mucosal H2S release in rats by upregulating mRNA and protein expression of cystathionine gamma lyase.

    Science.gov (United States)

    Mard, Seyyed Ali; Veisi, Ali; Ahangarpour, Akram; Gharib-Naseri, Mohammad Kazem

    2015-11-01

    It is well known that hydrogen sulfide (H2S) protects the gastric mucosa against gastric acid and other noxious stimulants by several mechanisms but until now the effect of gastric acid on H2S production has not been evaluated. This study was performed to determine the effect of basal and stimulated gastric acid secretion on mRNA and protein expression of cystathionine gamma lyase (CSE) and cystathionine beta synthase (CBS), and on mucosal release of H2S in rats. Seventy-two male rats were randomly assigned into 9 groups (8 in each)-control, distention, and pentagastrin-induced gastric acid secretion groups. The effects of 15% alcohol solution, propargylglycine (PAG), L-NAME, and pantoprazole were also investigated. Under anesthesia, animals underwent tracheostomy and midline laparotomy. A catheter was inserted into the stomach through the duodenum for gastric washout. At the end of the experiments, the animals were killed and the gastric mucosa was collected to measure H2S concentration and to quantify mRNA expression of CSE and CBS by quantitative real-time PCR, and expression of their proteins by western blot. Basal and stimulated gastric acid secretion increased mucosal levels of H2S, and mRNA and protein expression of CSE. Pantoprazole and L-NAME reversed H2S release and restored protein expression of CSE to the control level. Pantoprazole, but not propargylglycine, pretreatment inhibited the elevated level of protein expression of eNOS in response to distention-induced gastric acid secretion. Our findings indicated that NO mediated the stimulatory effect of gastric acid on H2S release and protein expression of CSE.

  10. Estrogen modulates the mRNA levels for cancellous bone protein of ovariectomized rats.

    Science.gov (United States)

    Salih, M A; Liu, C C; Arjmandi, B H; Kalu, D N

    1993-12-01

    This study was undertaken to examine the effects of ovariectomy and 17 beta-estradiol (E2) on the gene expression of type 1 collagen, osteocalcin and the protooncogen, c-myc, in cancellous bone. Female Sprague-Dawley rats, aged 95 days, were divided into 4 groups. Group 1 was sham operated and Groups 2-4 were ovariectomized. Groups 3 and 4 received daily injections of 160 ng and 1600 ng E2/kg body weight, respectively. Groups 1 and 2 received the solvent vehicle. All animals were sacrificed after 14 days. The femurs were dissected out and cancellous bone scraped from the distal metaphysis. RNA was isolated from the cancellous bone, immobilized on filters or size-fractionated by agarose gel electrophoresis and adsorbed on filters which were then hybridized with specific cDNA probes. Ovariectomy resulted in a significant increase in the mRNAs of type 1 collagen, osteocalcin and c-myc. The increase was suppressed in animals that received 17 beta-estradiol injections. In addition, ovariectomy caused the expected decrease in cancellous bone in the proximal tibia and increased osteoclast and osteoblast numbers. The ovariectomy-induced changes were prevented by 17 beta-estradiol administration. These findings suggest that the lack of ovarian hormones shortly after ovariectomy up-regulates and estrogen administration down-regulates the expression of important cancellous bone matrix proteins as well as the protooncogen, c-myc.

  11. Role of protein and mRNA oxidation in seed dormancy and germination

    Directory of Open Access Journals (Sweden)

    hayat eel-maarouf-bouteau

    2013-04-01

    Full Text Available Reactive oxygen species (ROS are key players in the regulation of seed germination and dormancy. Although their regulated accumulation is a prerequisite for germination, the cellular basis of their action remains unknown, but very challenging to elucidate due to the lack of specificity of these compounds that can potentially react with all biomolecules. Among these, nucleic acids and proteins are very prone to oxidative damage. RNA is highly sensitive to oxidation because of its single-stranded structure and the absence of a repair system. Oxidation of mRNAs induces their decay through processing bodies or results in the synthesis of aberrant proteins through altered translation. Depending on the oxidized amino acid, ROS damage of proteins can be irreversible (i.e. carbonylation thus triggering the degradation of the oxidized proteins by the cytosolic 20S proteasome or can be reversed through the action of thioredoxins, peroxiredoxins or glutaredoxins (cysteine oxidation or by methionine sulfoxide reductase (methionine oxidation. Seed dormancy alleviation in the dry state, referred to as after-ripening, requires both selective mRNA oxidation and protein carbonylation. Similarly, seed imbibition of non-dormant seeds is associated with targeted oxidation of a subset of proteins. Altogether, these specific features testify that such oxidative modifications play important role in commitment of the cellular functioning toward germination completion.

  12. The Rabies Virus L Protein Catalyzes mRNA Capping with GDP Polyribonucleotidyltransferase Activity.

    Science.gov (United States)

    Ogino, Minako; Ito, Naoto; Sugiyama, Makoto; Ogino, Tomoaki

    2016-05-21

    The large (L) protein of rabies virus (RABV) plays multiple enzymatic roles in viral RNA synthesis and processing. However, none of its putative enzymatic activities have been directly demonstrated in vitro. In this study, we expressed and purified a recombinant form of the RABV L protein and verified its guanosine 5'-triphosphatase and GDP polyribonucleotidyltransferase (PRNTase) activities, which are essential for viral mRNA cap formation by the unconventional mechanism. The RABV L protein capped 5'-triphosphorylated but not 5'-diphosphorylated RABV mRNA-start sequences, 5'-AACA(C/U), with GDP to generate the 5'-terminal cap structure G(5')ppp(5')A. The 5'-AAC sequence in the substrate RNAs was found to be strictly essential for RNA capping with the RABV L protein. Furthermore, site-directed mutagenesis showed that some conserved amino acid residues (G1112, T1170, W1201, H1241, R1242, F1285, and Q1286) in the PRNTase motifs A to E of the RABV L protein are required for cap formation. These findings suggest that the putative PRNTase domain in the RABV L protein catalyzes the rhabdovirus-specific capping reaction involving covalent catalysis of the pRNA transfer to GDP, thus offering this domain as a target for developing anti-viral agents.

  13. Downregulation of the microtubule associated protein tau impairs process outgrowth and myelin basic protein mRNA transport in oligodendrocytes.

    Science.gov (United States)

    Seiberlich, Veronika; Bauer, Nina G; Schwarz, Lisa; Ffrench-Constant, Charles; Goldbaum, Olaf; Richter-Landsberg, Christiane

    2015-09-01

    Oligodendrocytes, the myelin forming cells of the CNS, are characterized by their numerous membranous extensions, which enwrap neuronal axons and form myelin sheaths. During differentiation oligodendrocytes pass different morphological stages, downregulate the expression of the proteoglycan NG2, and acquire major myelin specific proteins, such as myelin basic proteins (MBP) and proteolipid protein. MBP mRNA is transported in RNA granules along the microtubules (MTs) to the periphery and translated locally. MTs participate in the elaboration and stabilization of the myelin forming extensions and are essential for cellular sorting processes. Their dynamic properties are regulated by microtubule associated proteins (MAPs). The MAP tau is present in oligodendrocytes and involved in the regulation and stabilization of the MT network. To further elucidate the functional significance of tau in oligodendrocytes, we have downregulated tau by siRNA technology and studied the effects on cell differentiation and neuron-glia contact formation. The data show that tau knockdown impairs process outgrowth and leads to a decrease in MBP expression. Furthermore, MBP mRNA transport to distant cellular extensions is impaired and cells remain in the NG2 stage. In myelinating cocultures with dorsal root ganglion neurons, oligodendrocyte precursor cells after tau miR RNA lentiviral knockdown develop into NG2 positive cells with very long and thin processes, contacting axons loosely, but fail to form internodes. This demonstrates that tau is important for MBP mRNA transport and involved in process formation. The disturbance of the balance of tau leads to abnormalities in oligodendrocyte differentiation, neuron-glia contact formation and the early myelination process.

  14. Influence of TGF-beta1 on the expression of BSP, DSP, TGF-beta1 receptor I and Smad proteins during reparative dentinogenesis.

    Science.gov (United States)

    Hwang, Yun-Chan; Hwang, In-Nam; Oh, Won-Mann; Park, Joo-Cheol; Lee, Dong-Seol; Son, Ho-Hyun

    2008-04-01

    Reparative dentin has a wide variety of manifestations ranging from a regular, tubular form to an irregular, atubular form. However, the characteristics of reparative dentin have not been clarified. This study hypothesized that the level of bone sialoprotein (BSP) expression will increase if the newly formed reparative dentin is bone-like but the dentin sialophosphoprotein (DSPP) level will decrease. In order to test this hypothesis, the expression of BSP and DSP was examined by immunohistochemistry and the expression of BSP was measured by in situ hybridization in an animal model. The pulps of 12 maxillary right first molars from twelve male rats were exposed and capped with MTA. In addition, in order to understand the role of transforming growth factor-beta 1 (TGF-beta1) during reparative dentinogenesis, the expression of BSP and DSPP mRNA was analyzed by RT-PCR in a human dental pulp cell culture, and the transforming growth factor-beta 1 receptors (TbetaRI) and Smad 2/3 were examined by immunofluorescence in an animal model. DSP was expressed in the normal odontoblasts and odontoblast-like cells of the reparative dentin. Interestingly, BSP was strongly expressed in the odontoblast-like cells of reparative dentin. The level of the TbetaRI and Smad 2/3 proteins was higher in the reparative dentin than in the normal dentin. TGF-beta1 up-regulated BSP in the human pulp cell cultures. This suggests that reparative dentin has both dentinogenic and osteogenic characteristics that are mediated by TGF-beta1.

  15. A-Raf kinase is a new interacting partner of protein kinase CK2 beta subunit

    DEFF Research Database (Denmark)

    Boldyreff, B; Issinger, O G

    1997-01-01

    In a search for protein kinase CK2 beta subunit binding proteins using the two-hybrid system, more than 1000 positive clones were isolated. Beside clones for the alpha' and beta subunit of CK2, there were clones coding for a so far unknown protein, whose partial cDNA sequence was already deposite...

  16. Changes of Survivin mRNA and Protein Expression during Paclitaxel Treatment in Breast Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    XIONG Huihua; YU Shiying; ZHUANG Liang; XIONG Hua

    2007-01-01

    In order to investigate the role of antiapoptosis gene, survivin in the resistance to palcitaxel, the expression of survivin mRNA and protein in the process of paclitaxel treatment in breast cancer cell line MCF-7 was detected. MCF-7 cells were incubated with paclitaxel at different concentrations. The growth inhibition rate of MCF-7 was investigated by tetrazolium bromide (MTT) colorimetry. The change of apoptosis was detected by Annexin-V/PI methods. The changes in the expression of survivin mRNA and protein were studied by reverse transcription polymerase chain reaction (RT-PCR) and Western-blot assay respectively. The growth inhibition rate of MCF-7 was increased in a concentration- and time-dependent manner. Paclitaxel of higher concentration could effectively induce apoptosis in MCF-7 cells after 48 h, while the expression of survivin was increased at early time (within 6 h) and decreased after 24 h regardless of treatment concentrations of paclitaxel. It suggested that tumor cells might evade the paclitaxel-induced cell cycle arrest and apoptosis by increasing the level of survivin at early treatment time.

  17. The structure of a ribosomal protein S8/spc operon mRNA complex.

    Science.gov (United States)

    Merianos, Helen J; Wang, Jimin; Moore, Peter B

    2004-06-01

    In bacteria, translation of all the ribosomal protein cistrons in the spc operon mRNA is repressed by the binding of the product of one of them, S8, to an internal sequence at the 5' end of the L5 cistron. The way in which the first two genes of the spc operon are regulated, retroregulation, is mechanistically distinct from translational repression by S8 of the genes from L5 onward. A 2.8 A resolution crystal structure has been obtained of Escherichia coli S8 bound to this site. Despite sequence differences, the structure of this complex is almost identical to that of the S8/helix 21 complex seen in the small ribosomal subunit, consistent with the hypothesis that autogenous regulation of ribosomal protein synthesis results from conformational similarities between mRNAs and rRNAs. S8 binding must repress the translation of its own mRNA by inhibiting the formation of a ribosomal initiation complex at the start of the L5 cistron.

  18. RAT GDNF GENE TRANSFECTION AND EXPRESSION OF ITS mRNA AND PROTEIN IN SCHWANN CELLS

    Institute of Scientific and Technical Information of China (English)

    平萍; 范志宏; 李青峰; 张涤生

    2004-01-01

    Objective To investigate the possibility of the transfection of glial-cell line derived neurotrophic factor (GDNF) gene into Schwann cells(SCs). Methods SCs cultures from sciatic nerves of neonatal rats were established. A recombinant retrovirus vector containing GDNF gene was constructed and transferred into SCs.Expression levels of GDNF mRNA and protein were respectively identified with reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry. Determination of GDNF synthesis rates from Retro. pLNCX2-GDNF-transduced SCs (GDNF-SCs) in vitro by enzyme-linked immunoassay sensitive assay ( ELISA ). Biololgical activity of conditioned medium from GENF-SCs was analysed by co-culture with rat motoneurons. Results Transfection of GDNF gene into SCs lead to significantly enhanced expression of GDNF mRNA and protein. The rate of GDNF secreted by GDNF-SCs was also enhanced(5. 1-fold), and more motoneurons survived co-cultured with conditioned medium of GNDF-SCs than with that of normal SCs. Conclusion GNDF gene transfection may be a better way to graft SCs promoting regeneration and repairing demyelination in PNS and CNS.

  19. Beta-secretase-cleaved amyloid precursor protein in Alzheimer brain: a morphologic study

    DEFF Research Database (Denmark)

    Sennvik, Kristina; Bogdanovic, N; Volkmann, Inga

    2004-01-01

    beta-amyloid (Abeta) is the main constituent of senile plaques seen in Alzheimer's disease. Abeta is derived from the amyloid precursor protein (APP) via proteolytic cleavage by proteases beta- and gamma-secretase. In this study, we examined content and localization of beta-secretase-cleaved APP...

  20. Protein-Ligand Docking Based on Beta-Shape

    Science.gov (United States)

    Kim, Chong-Min; Won, Chung-In; Kim, Jae-Kwan; Ryu, Joonghyun; Bhak, Jong; Kim, Deok-Soo

    Protein-ligand docking is to predict the location and orientation of a ligand with respect to a protein within its binding site, and has been known to be essential for the development of new drugs. The protein-ligand docking problem is usually formulated as an energy minimization problem to identify the docked conformation of the ligand. A ligand usually docks around a depressed region, called a pocket, on the surface of a protein. Presented in this paper is a docking method, called BetaDock, based on the newly developed geometric construct called the β-shape and the β-complex. To cope with the computational intractability, the global minimum of the potential energy function is searched using the genetic algorithm. The proposed algorithm first locates initial chromosomes at some locations within the pocket recognized according to the local shape of the β-shape. Then, the algorithm proceeds generations by taking advantage of powerful properties of the β-shape to achieve an extremely fast and good solution. We claim that the proposed method is much faster than other popular docking softwares including AutoDock.

  1. Beta-secretase-cleaved amyloid precursor protein in Alzheimer brain: a morphologic study

    DEFF Research Database (Denmark)

    Sennvik, Kristina; Bogdanovic, N; Volkmann, Inga

    2004-01-01

    beta-amyloid (Abeta) is the main constituent of senile plaques seen in Alzheimer's disease. Abeta is derived from the amyloid precursor protein (APP) via proteolytic cleavage by proteases beta- and gamma-secretase. In this study, we examined content and localization of beta-secretase-cleaved APP...... the beta-sAPP immunostaining to be stronger and more extensive in gray matter in Alzheimer disease (AD) cases than controls. The axonal beta-sAPP staining was patchy and unevenly distributed for the AD cases, indicating impaired axonal transport. beta-sAPP was also found surrounding senile plaques...

  2. Bcl-2/Bax protein and mRNA expression in yak (Bos grunniens) placentomes.

    Science.gov (United States)

    Fan, JiangFeng; Yu, SiJiu; Cui, Yan; Xu, Gengquan; Wang, Libin; Pan, Yangyang; He, Honghong

    2017-07-29

    Placental function is complex and influenced by various factors; furthermore, it depends on a delicate balance between cell proliferation, cell differentiation, and cell death. Bcl-2 and Bax proteins are key apoptosis regulators and are considered to play an important role in the maintenance of both dynamic balance and integrity of many tissues. Changes in Bcl-2 and Bax expressions have been described during different developmental stages in normal human placentas. Studies furthermore indicated several pathological placental changes to be related to abnormal Bcl-2 and Bax expressions. In the present study, we investigated both expression and distribution of Bcl-2 and Bax in yak placentas. For this, we collected placentas of 35 yaks at different stages of pregnancy as well as cotyledonary villi of four postpartum yaks. Protein and mRNA expressions of both Bcl-2 and Bax were investigated via immunohistochemistry, Western blot, and real-time PCR. Immunoreactive Bcl-2 protein was mainly localized near the fetal villous trophoblast at various gestational stages and post-partum. The Positive Index (PI) of Bcl-2 protein expression significantly decreased with increasing gestational age. Early during pregnancy (≤2 months), the Bax protein was widely distributed in the fetal villous trophoblast layer, the maternal caruncular crypt epithelium, and the stroma. Subsequently, the Bax protein distribution gradually concentrated in the fetal villous trophoblast layer. The staining intensity of Bax increased from the 3rd month to the prepartum of gestation. The PI reached a minimum of 9.4 ± 2.2 in fetal chorionic villi (FCV) and 1.3 ± 0.8 in maternal caruncular crypts (MCC) of the three months group. Both Bcl-2 and Bax had maximum immunoreactivity in the fetal villous trophoblast layer of placentas collected form postpartum yaks (with PIs of 36.6 ± 5.7 and 38.2 ± 4.8, respectively). Protein and mRNA expression of Bcl-2 and Bax investigated via Western blot and real

  3. Transforming growth factor-beta1 stimulates the production of insulin-like growth factor-I and insulin-like growth factor-binding protein-3 in human bone marrow stromal osteoblast progenitors

    DEFF Research Database (Denmark)

    Kveiborg, Marie; Flyvbjerg, Allan; Eriksen, E F

    2001-01-01

    While transforming growth factor-beta1 (TGF-beta1) regulates proliferation and differentiation of human osteoblast precursor cells, the mechanisms underlying these effects are not known. Several hormones and locally acting growth factors regulate osteoblast functions through changes in the insulin......-like growth factors (IGFs) and IGF-binding proteins (IGFBPs). Thus, we studied the effects of TGF-beta1 on IGFs and IGFBPs in human marrow stromal (hMS) osteoblast precursor cells. TGF-beta1 increased the steady-state mRNA level of IGF-I up to 8.5+/-0.6-fold (P...

  4. Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yeming; Opperman, Laura; Wickens, Marvin; Tanaka Hall, Traci M. (NIH); (UW)

    2011-11-02

    Caenorhabditis elegans fem-3 binding factor (FBF) is a founding member of the PUMILIO/FBF (PUF) family of mRNA regulatory proteins. It regulates multiple mRNAs critical for stem cell maintenance and germline development. Here, we report crystal structures of FBF in complex with 6 different 9-nt RNA sequences, including elements from 4 natural mRNAs. These structures reveal that FBF binds to conserved bases at positions 1-3 and 7-8. The key specificity determinant of FBF vs. other PUF proteins lies in positions 4-6. In FBF/RNA complexes, these bases stack directly with one another and turn away from the RNA-binding surface. A short region of FBF is sufficient to impart its unique specificity and lies directly opposite the flipped bases. We suggest that this region imposes a flattened curvature on the protein; hence, the requirement for the additional nucleotide. The principles of FBF/RNA recognition suggest a general mechanism by which PUF proteins recognize distinct families of RNAs yet exploit very nearly identical atomic contacts in doing so.

  5. Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yeming; Opperman, Laura; Wickens, Marvin; Tanaka Hall, Traci M.; (NIH); (UW)

    2010-08-19

    Caenorhabditis elegans fem-3 binding factor (FBF) is a founding member of the PUMILIO/FBF (PUF) family of mRNA regulatory proteins. It regulates multiple mRNAs critical for stem cell maintenance and germline development. Here, we report crystal structures of FBF in complex with 6 different 9-nt RNA sequences, including elements from 4 natural mRNAs. These structures reveal that FBF binds to conserved bases at positions 1-3 and 7-8. The key specificity determinant of FBF vs. other PUF proteins lies in positions 4-6. In FBF/RNA complexes, these bases stack directly with one another and turn away from the RNA-binding surface. A short region of FBF is sufficient to impart its unique specificity and lies directly opposite the flipped bases. We suggest that this region imposes a flattened curvature on the protein; hence, the requirement for the additional nucleotide. The principles of FBF/RNA recognition suggest a general mechanism by which PUF proteins recognize distinct families of RNAs yet exploit very nearly identical atomic contacts in doing so.

  6. Induction of complement proteins in a mouse model for cerebral microvascular A beta deposition.

    Science.gov (United States)

    Fan, Rong; DeFilippis, Kelly; Van Nostrand, William E

    2007-09-18

    The deposition of amyloid beta-protein (A beta) in cerebral vasculature, known as cerebral amyloid angiopathy (CAA), is a common pathological feature of Alzheimer's disease and related disorders. In familial forms of CAA single mutations in the A beta peptide have been linked to the increase of vascular A beta deposits accompanied by a strong localized activation of glial cells and elevated expression of neuroinflammatory mediators including complement proteins. We have developed human amyloid-beta precursor protein transgenic mice harboring two CAA A beta mutations (Dutch E693Q and Iowa D694N) that mimic the prevalent cerebral microvascular A beta deposition observed in those patients, and the Swedish mutations (K670N/M671L) to increase A beta production. In these Tg-SwDI mice, we have reported predominant fibrillar A beta along microvessels in the thalamic region and diffuse plaques in cortical region. Concurrently, activated microglia and reactive astrocytes have been detected primarily in association with fibrillar cerebral microvascular A beta in this model. Here we show that three native complement components in classical and alternative complement pathways, C1q, C3, and C4, are elevated in Tg-SwDI mice in regions rich in fibrillar microvascular A beta. Immunohistochemical staining of all three proteins was increased in thalamus, hippocampus, and subiculum, but not frontal cortex. Western blot analysis showed significant increases of all three proteins in the thalamic region (with hippocampus) as well as the cortical region, except C3 that was below detection level in cortex. Also, in the thalamic region (with hippocampus), C1q and C3 mRNAs were significantly up-regulated. These complement proteins appeared to be expressed largely by activated microglial cells associated with the fibrillar microvascular A beta deposits. Our findings demonstrate that Tg-SwDI mice exhibit elevated complement protein expression in response to fibrillar vascular A beta deposition

  7. Interleukin-1beta induced changes in the protein expression of rat islets: a computerized database

    DEFF Research Database (Denmark)

    Andersen, H U; Fey, S J; Larsen, Peter Mose

    1997-01-01

    Insulin-dependent diabetes mellitus is caused by an autoimmune destruction of the beta-cells in the islets of Langerhans. The cytokine interleukin 1 inhibits insulin release and is selectively cytotoxic to beta-cells in isolated pancreatic rat islets. The antigen(s) triggering the immune response...... as well as the intracellular mechanisms of action of interleukin 1-mediated beta-cell cytotoxicity are unknown. However, previous studies have found an association of beta-cell destruction with alterations in protein synthesis. Thus, two-dimensional (2-D) gel electrophoresis of pancreatic islet proteins......% of %IOD was 45.7% in the NEPHGE gels. Addition of interleukin-1beta (IL-1beta) to the cultures resulted in statistically significant modulation or de novo synthesis of 105 proteins in the 10% gels. In conclusion, we present the first 10% and 15% acrylamide 2-D gel protein databases of neonatal rat islets...

  8. Translation of myelin basic protein mRNA in oligodendrocytes is regulated by integrin activation and hnRNP-K

    DEFF Research Database (Denmark)

    Laursen, Lisbeth Schmidt; Chan, Colin W; ffrench-Constant, Charles

    2011-01-01

    Myelination in the central nervous system provides a unique example of how cells establish asymmetry. The myelinating cell, the oligodendrocyte, extends processes to and wraps multiple axons of different diameter, keeping the number of wraps proportional to the axon diameter. Local regulation...... translation of a key sheath protein, myelin basic protein (MBP), by reversing the inhibitory effect of the mRNA 3′UTR. During oligodendrocyte differentiation and myelination α6β1-integrin interacts with hnRNP-K, an mRNA-binding protein, which binds to MBP mRNA and translocates from the nucleus to the myelin...

  9. Conserved CPEs in the p53 3' untranslated region influence mRNA stability and protein synthesis

    DEFF Research Database (Denmark)

    Rosenstierne, Maiken W; Vinther, Jeppe; Mittler, Gerhard;

    2008-01-01

    CaT skin and MCF-7 breast cancer cell lines were established. Quantitative PCR and an enzymatic assay were used to quantify the reporter mRNA and protein levels, respectively. Proteins binding to the CPEs were identified by RNA-immunoprecipitation (IP) and quantitative mass spectroscopy. RESULTS: The wild-type...

  10. Changes in myocardial beta1-adrenergic receptor and stimulatory G-protein gene expression after chronic treatment with doxorubicin in rat.

    Science.gov (United States)

    Kizaki, Keiichiro; Akatsuka, Keiko; Momozaki, Masami; Fujimori, Yuuki; Uchide, Tsuyoshi; Temma, Kyosuke; Hara, Yukio

    2004-08-01

    The gene expression of beta(1)-adrenergic receptor (beta(1)AR) and stimulatory G-protein Gsalpha in ventricle after chronic treatment with doxorubicin (DOX) in rat was investigated. The rats were treated with DOX in a dose of 2.5 mg/kg once a week for 5 weeks, the cumulative dose being 12.5 mg/kg. Two weeks after the last injection, the positive inotropic effect of isoproterenol was noticeably decreased in left atrial muscle preparations isolated from DOX-treated rats. Northern blot hybridization showed that the mRNA transcripts of beta(1)AR and Gsalpha, important signal transduction elements for regulating heart rate and contractility, were significantly decreased in the ventricle of DOX-treated rats. Thus, chronic treatment with DOX decreases the gene expression levels of myocardial beta(1)AR and Gsalpha.

  11. Arabidopsis mRNA secondary structure correlates with protein function and domains

    Science.gov (United States)

    Vandivier, Lee E.; Li, Fan; Zheng, Qi; Willmann, Matthew R.; Chen, Ying; Gregory, Brian D.

    2013-01-01

    RNAs fold into intricate structures that are determined by specific base pairing interactions encoded within their primary sequences. Recently, a number of transcriptome-wide studies have suggested that RNA secondary structure is a potent cis-acting regulator of numerous post-transcriptional processes in viruses and eukaryotes. However, the need for experimentally-based structure determination methods has not been well addressed. Here, we show that the regulatory significance of Arabidopsis RNA secondary structure is revealed specifically through high-throughput, sequencing-based, structure mapping data, not by computational prediction. Additionally, we find that transcripts with similar levels of secondary structure in their UTRs (5' or 3') or CDS tend to encode proteins with coherent functions. Finally, we reveal that portions of mRNAs encoding predicted protein domains are significantly more structured than those specifying inter-domain regions. In total, our findings show the utility of high-throughput, sequencing-based, structure-mapping approaches and suggest that mRNA folding regulates protein maturation and function. PMID:23603972

  12. Expression of Beta-Catenin and APC Protein in Ovarian Epithelial Tumor and Its Implication

    Institute of Scientific and Technical Information of China (English)

    LIN Xiao; LI Yu; MI Can

    2007-01-01

    Objective: To investigate the expression of beta-catenin, APC protein and its implication in ovarian epithelial tumor. Methods: Immunohistochemical staining with SP method was conducted to determine the expression of beta-catenin and APC protein in 48 cases of ovarian epithelial tumor. Results: The abnormal expression rates of beta-catenin in ovarian malignant and borderline epithelial tumors were higher than that in benign epithelial tumors. The expression of APC protein in benign epithelial tumors was significantly greater than that in malignant epithelial tumors. A significant negative correlation was found between beta-catenin and APC protein in ovarian epithelial tumors. Conclusion: Beta-catenin and APC protein have important effect on pathogenesis and development of ovarian epithelial tumors.

  13. Chemokines, macrophage inflammatory protein-2 and stromal cell-derived factor-1{alpha}, suppress amyloid {beta}-induced neurotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Raman, Dayanidhi; Milatovic, Snjezana-Zaja [Department of Cancer Biology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States); Milatovic, Dejan [Department of Pediatrics/Pediatric Toxicology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States); Splittgerber, Ryan [Department of Cancer Biology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States); Fan, Guo-Huang [Department of Neurobiology and Neurotoxicology, Meharry Medical College, Nashville, TN 37221 (United States); Richmond, Ann, E-mail: ann.richmond@vanderbilt.edu [VA Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States)

    2011-11-15

    Alzheimer's disease (AD) is characterized by a progressive cognitive decline and accumulation of neurotoxic oligomeric peptides amyloid-{beta} (A{beta}). Although the molecular events are not entirely known, it has become evident that inflammation, environmental and other risk factors may play a causal, disruptive and/or protective role in the development of AD. The present study investigated the ability of the chemokines, macrophage inflammatory protein-2 (MIP-2) and stromal cell-derived factor-1{alpha} (SDF-1{alpha}), the respective ligands for chemokine receptors CXCR2 and CXCR4, to suppress A{beta}-induced neurotoxicity in vitro and in vivo. Pretreatment with MIP-2 or SDF-1{alpha} significantly protected neurons from A{beta}-induced dendritic regression and apoptosis in vitro through activation of Akt, ERK1/2 and maintenance of metalloproteinase ADAM17 especially with SDF-1{alpha}. Intra-cerebroventricular (ICV) injection of A{beta} led to reduction in dendritic length and spine density of pyramidal neurons in the CA1 area of the hippocampus and increased oxidative damage 24 h following the exposure. The A{beta}-induced morphometric changes of neurons and increase in biomarkers of oxidative damage, F{sub 2}-isoprostanes, were significantly inhibited by pretreatment with the chemokines MIP-2 or SDF-1{alpha}. Additionally, MIP-2 or SDF-1{alpha} was able to suppress the aberrant mislocalization of p21-activated kinase (PAK), one of the proteins involved in the maintenance of dendritic spines. Furthermore, MIP-2 also protected neurons against A{beta} neurotoxicity in CXCR2-/- mice, potentially through observed up regulation of CXCR1 mRNA. Understanding the neuroprotective potential of chemokines is crucial in defining the role for their employment during the early stages of neurodegeneration. -- Research highlights: Black-Right-Pointing-Pointer Neuroprotective ability of the chemokines MIP2 and CXCL12 against A{beta} toxicity. Black-Right-Pointing-Pointer MIP

  14. A Physiological Role for Amyloid Beta Protein: Enhancement of Learning and Memory

    OpenAIRE

    John Morley; Susan Farr; William Banks; Johnson, Steven N.; Yamada, Kelvin A.; Lin Xu

    2008-01-01

    Amyloid beta protein (A[beta]) is well recognized as having a significant role in the pathogenesis of Alzheimer's disease (AD). The reason for the presence of A[beta] and its physiological role in non-disease states is not clear. In these studies, low doses of A[beta] enhanced memory retention in two memory tasks and enhanced acetylcholine production in the hippocampus _in vivo_. We then tested whether endogenous A[beta] has a role in learning and memory in young, cognitively intact mice...

  15. Changes of Expression of Stretch-activated Potassium Channel TREK-1 mRNA and Protein in Hypertrophic Myocardium

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The expression of stretch-activated potassium channel TREK-1 mRNA and protein of hypertrophic myocardium was measured. Using a model of hypertrophy induced by coarctation of abdominal aorta in male Wistar rats, the expression of TREK-1 mRNA and protein was detected by using semi quantitative RT PCR and Western blot respectively. At 4th and 8th week after constriction of the abdominal aorta, rats developed significant left ventricular hypertrophy. As compared to sham operated group, stretch activated potassium channel TREK-1 mRNA was strongly expressed and protein was up regulated in operation groups (P<0.05). It was concluded that the expression of TREK 1 was up regulated in hypertrophic myocardium induced by chronic pressure overload in Wistar rats.

  16. Novel RNA- and FMRP-binding protein TRF2-S regulates axonal mRNA transport and presynaptic plasticity.

    Science.gov (United States)

    Zhang, Peisu; Abdelmohsen, Kotb; Liu, Yong; Tominaga-Yamanaka, Kumiko; Yoon, Je-Hyun; Ioannis, Grammatikakis; Martindale, Jennifer L; Zhang, Yongqing; Becker, Kevin G; Yang, In Hong; Gorospe, Myriam; Mattson, Mark P

    2015-11-20

    Despite considerable evidence that RNA-binding proteins (RBPs) regulate mRNA transport and local translation in dendrites, roles for axonal RBPs are poorly understood. Here we demonstrate that a non-telomeric isoform of telomere repeat-binding factor 2 (TRF2-S) is a novel RBP that regulates axonal plasticity. TRF2-S interacts directly with target mRNAs to facilitate their axonal delivery. The process is antagonized by fragile X mental retardation protein (FMRP). Distinct from the current RNA-binding model of FMRP, we show that FMRP occupies the GAR domain of TRF2-S protein to block the assembly of TRF2-S-mRNA complexes. Overexpressing TRF2-S and silencing FMRP promotes mRNA entry to axons and enhances axonal outgrowth and neurotransmitter release from presynaptic terminals. Our findings suggest a pivotal role for TRF2-S in an axonal mRNA localization pathway that enhances axon outgrowth and neurotransmitter release.

  17. Evolutionary Conservation and Diversification of Puf RNA Binding Proteins and Their mRNA Targets.

    Science.gov (United States)

    Hogan, Gregory J; Brown, Patrick O; Herschlag, Daniel

    2015-01-01

    chain (ETC) complex I as well as hundreds of other mRNAs with nonmitochondrial functions. The many concerted and conserved changes in the RNA targets of Puf proteins strongly support an extensive role of RNA binding proteins in coordinating gene expression, as originally proposed by Keene. Rewiring of Puf-coordinated mRNA targets and transcriptional control of the same genes occurred at different points in evolution, suggesting that there have been distinct adaptations via RNA binding proteins and transcription factors. The changes in Puf targets and in the Puf proteins indicate an integral involvement of RNA binding proteins and their RNA targets in the adaptation, reprogramming, and function of gene expression.

  18. Evolutionary Conservation and Diversification of Puf RNA Binding Proteins and Their mRNA Targets.

    Directory of Open Access Journals (Sweden)

    Gregory J Hogan

    transport chain (ETC complex I as well as hundreds of other mRNAs with nonmitochondrial functions. The many concerted and conserved changes in the RNA targets of Puf proteins strongly support an extensive role of RNA binding proteins in coordinating gene expression, as originally proposed by Keene. Rewiring of Puf-coordinated mRNA targets and transcriptional control of the same genes occurred at different points in evolution, suggesting that there have been distinct adaptations via RNA binding proteins and transcription factors. The changes in Puf targets and in the Puf proteins indicate an integral involvement of RNA binding proteins and their RNA targets in the adaptation, reprogramming, and function of gene expression.

  19. Estrogen and exercise interact to regulate brain-derived neurotrophic factor mRNA and protein expression in the hippocampus.

    Science.gov (United States)

    Berchtold, N C; Kesslak, J P; Pike, C J; Adlard, P A; Cotman, C W

    2001-12-01

    We investigated the possibility that estrogen and exercise interact in the hippocampus and regulate brain-derived neurotrophic factor (BDNF), a molecule increasingly recognized for its role in plasticity and neuron function. An important aspect of this study is to examine the effect of different time intervals between estrogen loss and estrogen replacement intervention. We demonstrate that in the intact female rat, physical activity increases hippocampal BDNF mRNA and protein levels. However, the exercise effect on BDNF up-regulation is reduced in the absence of estrogen, in a time-dependent manner. In addition, voluntary activity itself is stimulated by the presence of estrogen. In exercising animals, estrogen deprivation reduced voluntary activity levels, while estrogen replacement restored activity to normal levels. In sedentary animals, estrogen deprivation (ovariectomy) decreased baseline BDNF mRNA and protein, which were restored by estrogen replacement. Despite reduced activity levels in the ovariectomized condition, exercise increased BDNF mRNA levels in the hippocampus after short-term (3 weeks) estrogen deprivation. However, long-term estrogen-deprivation blunted the exercise effect. After 7 weeks of estrogen deprivation, exercise alone no longer affected either BDNF mRNA or protein levels. However, exercise in combination with long-term estrogen replacement increased BDNF protein above the effects of estrogen replacement alone. Interestingly, protein levels across all conditions correlated most closely with mRNA levels in the dentate gyrus, suggesting that expression of mRNA in this hippocampal region may be the major contributor to the hippocampal BDNF protein pool. The interaction of estrogen, physical activity and hippocampal BDNF is likely to be an important issue for maintenance of brain health, plasticity and general well-being, particularly in women.

  20. Hypothyroidism decreases proinsulin gene expression and the attachment of its mRNA and eEF1A protein to the actin cytoskeleton of INS-1E cells

    Directory of Open Access Journals (Sweden)

    F. Goulart-Silva

    2011-10-01

    Full Text Available The actions of thyroid hormone (TH on pancreatic beta cells have not been thoroughly explored, with current knowledge being limited to the modulation of insulin secretion in response to glucose, and beta cell viability by regulation of pro-mitotic and pro-apoptotic factors. Therefore, the effects of TH on proinsulin gene expression are not known. This led us to measure: a proinsulin mRNA expression, b proinsulin transcripts and eEF1A protein binding to the actin cytoskeleton, c actin cytoskeleton arrangement, and d proinsulin mRNA poly(A tail length modulation in INS-1E cells cultured in different media containing: i normal fetal bovine serum - FBS (control; ii normal FBS plus 1 µM or 10 nM T3, for 12 h, and iii FBS depleted of TH for 24 h (Tx. A decrease in proinsulin mRNA content and attachment to the cytoskeleton were observed in hypothyroid (Tx beta cells. The amount of eEF1A protein anchored to the cytoskeleton was also reduced in hypothyroidism, and it is worth mentioning that eEF1A is essential to attach transcripts to the cytoskeleton, which might modulate their stability and rate of translation. Proinsulin poly(A tail length and cytoskeleton arrangement remained unchanged in hypothyroidism. T3 treatment of control cells for 12 h did not induce any changes in the parameters studied. The data indicate that TH is important for proinsulin mRNA expression and translation, since its total amount and attachment to the cytoskeleton are decreased in hypothyroid beta cells, providing evidence that effects of TH on carbohydrate metabolism also include the control of proinsulin gene expression.

  1. The function of the inner nuclear envelope protein SUN1 in mRNA export is regulated by phosphorylation.

    Science.gov (United States)

    Li, Ping; Stumpf, Maria; Müller, Rolf; Eichinger, Ludwig; Glöckner, Gernot; Noegel, Angelika A

    2017-08-22

    SUN1, a component of the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex, functions in mammalian mRNA export through the NXF1-dependent pathway. It associates with mRNP complexes by direct interaction with NXF1. It also binds to the NPC through association with the nuclear pore component Nup153, which is involved in mRNA export. The SUN1-NXF1 association is at least partly regulated by a protein kinase C (PKC) which phosphorylates serine 113 (S113) in the N-terminal domain leading to reduced interaction. The phosphorylation appears to be important for the SUN1 function in nuclear mRNA export since GFP-SUN1 carrying a S113A mutation was less efficient in restoring mRNA export after SUN1 knockdown as compared to the wild type protein. By contrast, GFP-SUN1-S113D resembling the phosphorylated state allowed very efficient export of poly(A)+RNA. Furthermore, probing a possible role of the LINC complex component Nesprin-2 in this process we observed impaired mRNA export in Nesprin-2 knockdown cells. This effect might be independent of SUN1 as expression of a GFP tagged SUN-domain deficient SUN1, which no longer can interact with Nesprin-2, did not affect mRNA export.

  2. Increase in uncoupling protein-2 mRNA expression by BRL49653 and bromopalmitate in human adipocytes.

    Science.gov (United States)

    Viguerie-Bascands, N; Saulnier-Blache, J S; Dandine, M; Dauzats, M; Daviaud, D; Langin, D

    1999-03-05

    Uncoupling protein-2 (UCP2) is a novel mitochondrial protein that may be involved in the control of energy expenditure. We have previously reported an upregulation of adipose tissue UCP2 mRNA expression during fasting in humans. Analysis of changes in metabolic parameters suggested that fatty acids may be associated with the increased UCP2 mRNA level. Culture of human adipose tissue explants was used to study in vitro regulation of adipocyte UCP2 gene expression. A 48-h treatment with BRL49653 and bromopalmitate, two potent activators of PPARgamma, resulted in a dose-dependent increase in UCP2 mRNA levels. The induction by BRL49653 was rapid (from 6 h) and maintained up to 5 days. TNFalpha provoked a 2-fold decrease in UCP2 mRNA levels. Human recombinant leptin did not affect UCP2 mRNA expression. The data support the hypothesis that fatty acids are involved in the control of adipocyte UCP2 mRNA expression in humans. Copyright 1999 Academic Press.

  3. Molecular simulations of beta-amyloid protein near hydrated lipids (PECASE).

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, Aidan Patrick; Han, Kunwoo (Texas A& M University, College Station, TX); Ford, David M. (Texas A& M University, College Station, TX)

    2005-12-01

    We performed molecular dynamics simulations of beta-amyloid (A{beta}) protein and A{beta} fragment(31-42) in bulk water and near hydrated lipids to study the mechanism of neurotoxicity associated with the aggregation of the protein. We constructed full atomistic models using Cerius2 and ran simulations using LAMMPS. MD simulations with different conformations and positions of the protein fragment were performed. Thermodynamic properties were compared with previous literature and the results were analyzed. Longer simulations and data analyses based on the free energy profiles along the distance between the protein and the interface are ongoing.

  4. An essential nuclear protein in trypanosomes is a component of mRNA transcription/export pathway.

    Directory of Open Access Journals (Sweden)

    Mariana Serpeloni

    Full Text Available In eukaryotic cells, different RNA species are exported from the nucleus via specialized pathways. The mRNA export machinery is highly integrated with mRNA processing, and includes a different set of nuclear transport adaptors as well as other mRNA binding proteins, RNA helicases, and NPC-associated proteins. The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, a widespread and neglected human disease which is endemic to Latin America. Gene expression in Trypanosoma has unique characteristics, such as constitutive polycistronic transcription of protein-encoding genes and mRNA processing by trans-splicing. In general, post-transcriptional events are the major points for regulation of gene expression in these parasites. However, the export pathway of mRNA from the nucleus is poorly understood. The present study investigated the function of TcSub2, which is a highly conserved protein ortholog to Sub2/ UAP56, a component of the Transcription/Export (TREX multiprotein complex connecting transcription with mRNA export in yeast/human. Similar to its orthologs, TcSub2 is a nuclear protein, localized in dispersed foci all over the nuclei -except the fibrillar center of nucleolus- and at the interface between dense and non-dense chromatin areas, proposing the association of TcSub2 with transcription/processing sites. These findings were analyzed further by BrUTP incorporation assays and confirmed that TcSub2 is physically associated with active RNA polymerase II (RNA pol II, but not RNA polymerase I (RNA pol I or Spliced Leader (SL transcription, demonstrating participation particularly in nuclear mRNA metabolism in T. cruzi. The double knockout of the TcSub2 gene is lethal in T. cruzi, suggesting it has an essential function. Alternatively, RNA interference assays were performed in Trypanosoma brucei. It allowed demonstrating that besides being an essential protein, its knockdown causes mRNA accumulation in the nucleus and

  5. Contact prediction for beta and alpha-beta proteins using integer linear optimization and its impact on the first principles 3D structure prediction method ASTRO-FOLD.

    Science.gov (United States)

    Rajgaria, R; Wei, Y; Floudas, C A

    2010-06-01

    An integer linear optimization model is presented to predict residue contacts in beta, alpha + beta, and alpha/beta proteins. The total energy of a protein is expressed as sum of a C(alpha)-C(alpha) distance dependent contact energy contribution and a hydrophobic contribution. The model selects contact that assign lowest energy to the protein structure as satisfying a set of constraints that are included to enforce certain physically observed topological information. A new method based on hydrophobicity is proposed to find the beta-sheet alignments. These beta-sheet alignments are used as constraints for contacts between residues of beta-sheets. This model was tested on three independent protein test sets and CASP8 test proteins consisting of beta, alpha + beta, alpha/beta proteins and it was found to perform very well. The average accuracy of the predictions (separated by at least six residues) was approximately 61%. The average true positive and false positive distances were also calculated for each of the test sets and they are 7.58 A and 15.88 A, respectively. Residue contact prediction can be directly used to facilitate the protein tertiary structure prediction. This proposed residue contact prediction model is incorporated into the first principles protein tertiary structure prediction approach, ASTRO-FOLD. The effectiveness of the contact prediction model was further demonstrated by the improvement in the quality of the protein structure ensemble generated using the predicted residue contacts for a test set of 10 proteins.

  6. Expression of DNA Repair Enzyme hMTH1 mRNA and Protein in Hepatocellular Carcinoma

    Institute of Scientific and Technical Information of China (English)

    ZHOU Hejun; CHENG Bin; LIN Jusheng

    2005-01-01

    To study the expression of DNA repair enzyme hMTH1 mRNA and protein in hepatocellular carcinoma (HCC) tissues, tissues adjacent to the cancers, normal liver cells and hepatoma cell lines, and to investigate their function in the progress of HCC, semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) was employed to examine the expression of hMTH1 mRNA in matched HCC tissues (HT)/surrounding tissues (HST) of HCC, normal liver cell L02 and hepatoma cell lines SMMC7721, HepG2. hMTH1 protein was detected in corresponding HT as well as their HST by immunohistochemistry. Our results showed that the expression level of hMTH1 mRNA in HT was higher than that in HST (t=2.424, P <0.05). The expression level of hMTH1 mRNA in two hepatoma cell lines was higher than that in normal liver cell line (F=6.810, P <0.01). The expression of hMTH1 mRNA in SMMC7721 was similar to that in HepG2. hMTH1 protein was 88.2 % (15 of 17) positive in HT and 82.4 % (14 of 17) in HST. The protein level of hMTH1 in HT was correspondingly higher than in their HST (t=2.618,P<0.05). It is concluded that hMTH1 mRNA and protein were over-expressed in HCC and hepatoma cell lines. It may be one of the key events during the carcinogenesis,progression of HCC and may promote the malignant growth. These results suggest that hMTH1 plays a role in HCC and may be a candidate marker for the diagnosis of HCC.

  7. Extracellular heat shock protein HSP90{beta} secreted by MG63 osteosarcoma cells inhibits activation of latent TGF-{beta}1

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Shigeki [Functional Genomics Section, Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD (United States); Kulkarni, Ashok B., E-mail: ak40m@nih.gov [Functional Genomics Section, Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD (United States)

    2010-07-30

    Transforming growth factor-beta 1 (TGF-{beta}1) is secreted as a latent complex, which consists of latency-associated peptide (LAP) and the mature ligand. The release of the mature ligand from LAP usually occurs through conformational change of the latent complex and is therefore considered to be the first step in the activation of the TGF-{beta} signaling pathway. So far, factors such as heat, pH changes, and proteolytic cleavage are reportedly involved in this activation process, but the precise molecular mechanism is still far from clear. Identification and characterization of the cell surface proteins that bind to LAP are important to our understanding of the latent TGF-{beta} activation process. In this study, we have identified heat shock protein 90 {beta} (HSP90{beta}) from the cell surface of the MG63 osteosarcoma cell line as a LAP binding protein. We have also found that MG63 cells secrete HSP90{beta} into extracellular space which inhibits the activation of latent TGF-{beta}1, and that there is a subsequent decrease in cell proliferation. TGF-{beta}1-mediated stimulation of MG63 cells resulted in the increased cell surface expression of HSP90{beta}. Thus, extracellular HSP90{beta} is a negative regulator for the activation of latent TGF-{beta}1 modulating TGF-{beta} signaling in the extracellular domain. -- Research highlights: {yields} Transforming growth factor-beta 1 (TGF-{beta}1) is secreted as a latent complex. {yields} This complex consists of latency-associated peptide (LAP) and the mature ligand. {yields} The release of the mature ligand from LAP is the first step in TGF-{beta} activation. {yields} We identified for the first time a novel mechanism for this activation process. {yields} Heat shock protein 90 {beta} is discovered as a negative regulator for this process.

  8. Amyloid Beta-Protein and Neural Network Dysfunction

    Directory of Open Access Journals (Sweden)

    Fernando Peña-Ortega

    2013-01-01

    Full Text Available Understanding the neural mechanisms underlying brain dysfunction induced by amyloid beta-protein (Aβ represents one of the major challenges for Alzheimer’s disease (AD research. The most evident symptom of AD is a severe decline in cognition. Cognitive processes, as any other brain function, arise from the activity of specific cell assemblies of interconnected neurons that generate neural network dynamics based on their intrinsic and synaptic properties. Thus, the origin of Aβ-induced cognitive dysfunction, and possibly AD-related cognitive decline, must be found in specific alterations in properties of these cells and their consequences in neural network dynamics. The well-known relationship between AD and alterations in the activity of several neural networks is reflected in the slowing of the electroencephalographic (EEG activity. Some features of the EEG slowing observed in AD, such as the diminished generation of different network oscillations, can be induced in vivo and in vitro upon Aβ application or by Aβ overproduction in transgenic models. This experimental approach offers the possibility to study the mechanisms involved in cognitive dysfunction produced by Aβ. This type of research may yield not only basic knowledge of neural network dysfunction associated with AD, but also novel options to treat this modern epidemic.

  9. The Neuroprotective Functions of Transforming Growth Factor Beta Proteins

    Directory of Open Access Journals (Sweden)

    Gábor Lovas

    2012-07-01

    Full Text Available Transforming growth factor beta (TGF-β proteins are multifunctional cytokines whose neural functions are increasingly recognized. The machinery of TGF-β signaling, including the serine kinase type transmembrane receptors, is present in the central nervous system. However, the 3 mammalian TGF-β subtypes have distinct distributions in the brain suggesting different neural functions. Evidence of their involvement in the development and plasticity of the nervous system as well as their functions in peripheral organs suggested that they also exhibit neuroprotective functions. Indeed, TGF-β expression is induced following a variety of types of brain tissue injury. The neuroprotective function of TGF-βs is most established following brain ischemia. Damage in experimental animal models of global and focal ischemia was shown to be attenuated by TGF-βs. In addition, support for their neuroprotective actions following trauma, sclerosis multiplex, neurodegenerative diseases, infections, and brain tumors is also accumulating. The review will also describe the potential mechanisms of neuroprotection exerted by TGF-βs including anti-inflammatory, -apoptotic, -excitotoxic actions as well as the promotion of scar formation, angiogenesis, and neuroregeneration. The participation of these mechanisms in the neuroprotective effects of TGF-βs during different brain lesions will also be discussed.

  10. beta-Scission of C-3 (beta-carbon) alkoxyl radicals on peptides and proteins

    DEFF Research Database (Denmark)

    Headlam, H A; Mortimer, A; Easton, C J

    2000-01-01

    of new reactive groups, including hydroperoxides. These processes can result in the loss of structural or enzymatic activity. Backbone fragmentation is known to occur via a number of mechanisms, most of which involve hydrogen abstraction from the alpha-carbon site on the backbone. In this study, we...... demonstrate that initial attack at a side chain site, the beta-position (C-3), can give rise to formation of alpha-carbon radicals, and hence backbone cleavage, via the formation and subsequent beta-scission of C-3 alkoxyl radicals. This beta-scission reaction is rapid (k estimated to be >10(7) s(-)(1)) even...

  11. How is mRNA expression predictive for protein expression? A correlation study on human circulating monocytes

    Institute of Scientific and Technical Information of China (English)

    Yanfang Guo; Yuan Chen; Hui Jiang; Lijun Tan; Jingyun Xie; Xuezhen Zhu; Songping Liang; Hongwen Deng; Peng Xiao; Shufeng Lei; Feiyan Deng; Gary Guishan Xiao; Yaozhong Liu; Xiangding Chen; Liming Li; Shan Wu

    2008-01-01

    A key assumption in studying mRNA expression is that it is informative in the prediction of protein expression. However,only limited studies have explored the mRNA-protein expression correlation in yeast or human tissues and the results have been relatively inconsistent. We carried out correlation analyses on mRNA-protein expressions in freshly isolated human circulating monocytes from 30 unrelated women. The expressed proteins for 71 genes were quantified and identified by 2-D electrophoresis coupled with mass spectrometry. The corresponding mRNA expressions were quantified by Affymetrix gene chips. Significant correlation (r=0.235, P<0.0001) was observed for the whole dataset including all studied genes and all samples. The correlations varied in different biological categories of gene ontology. For example, the highest correlation was achieved for genes of the extracellular region in terms of cellular component (r=0.643, P<0.0001) and the lowest correlation was obtained for genes of regulation (r=0.099, P=0.213) in terms of biological process. In the genome, half of the samples showed significant positive correlation for the 71 genes and significant correlation was found between the average mRNA and the average protein expression levels in all samples (r=0.296, P<0.01). However, at the study group level, only five studied genes had significant positive correlation across all the samples. Our results showed an overall positive correlation between mRNA and protein expression levels.However, the moderate and varied correlations suggest that mRNA expression might be sometimes useful, but certainly far from perfect, in predicting protein expression levels.

  12. Expression patterns of ubiquitin, heat shock protein 70, alpha-actin and beta-actin over the molt cycle in the abdominal muscle of marine shrimp Litopenaeus vannamei.

    Science.gov (United States)

    Cesar, Jose Renato O; Yang, Jinzeng

    2007-05-01

    Crustacean muscle growth is discontinuous due to molt cycle. To characterize molt-related gene expression patterns, we studied the mRNA levels of molecular chaperone-ubiquitin and heat shock protein 70 (Hsp 70) in comparison with muscle protein alpha-actin and beta-actin in marine shrimp Litopenaeus vannamei. Total RNA from abdominal muscle was isolated from 3-month-old animals in six different molt stages. The mRNA levels of target genes were detected by reverse-transcriptase-multiplex PCR and expressed as the ratio to elongation factor-1alpha. Ubiquitin mRNA levels were relatively steady over all stages of the molt cycle. Hsp70 levels were not detectable in early postmolt and late premolt stages, but showed a progressive increase from late postmolt to intermolt stages. Expression levels of alpha-actin gene were lower during postmolt, reached a plateau in intermolt and remained relatively high in premolt stage. Levels of beta-actin increased progressively from postmolt to intermolt, reaching a maximum value in premolt. Therefore, the mRNAs encoding for ubiquitin and Hsp 70 in abdominal muscle did not increase significantly in premolt stages, which is typically associated with claw muscle degradation. Muscle structural alpha-actin and cytoskeletal beta-actin were increased during intermolt and premolt stages, suggesting high muscle growth during these stages in the abdominal muscle of the L. vannamei.

  13. Increased plasma amyloid beta protein 1-42 levels in Down syndrome.

    Science.gov (United States)

    Mehta, P D; Dalton, A J; Mehta, S P; Kim, K S; Sersen, E A; Wisniewski, H M

    1998-01-23

    Amyloid beta protein 1-40 (A beta40) and A beta42 levels were quantitated in plasma from 43 persons with Down syndrome (DS; 26-68 years of age), 43 age-matched normal controls, and 19 non-DS mentally retarded (MR) persons (26-91 years of age) by using a sandwich enzyme linked immunosorbent assay. A beta40 levels were higher in DS and MR than controls, but were similar between DS and MR groups. A beta42 levels were higher in DS than controls or MR persons. The ratios of A beta42/A beta40 were higher in DS than controls or MR persons. The findings are consistent with those seen in DS brains.

  14. Expressions of Beta-Catenin, SUFU and VEGFR-2 Proteins in Medulloblastoma

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xiong; ZHANG Hong-mei; LI Yu; MI Can

    2007-01-01

    Objective: to investigate the expressions of beta-catenin, SUFU and VEGFR-2 proteins in medulloblastoma. Methods: Immunohistochemical staining with SP method was conducted to determine the expressions of beta-catenin, SUFU and VEGFR-2 in 33 cases of medulloblastoma and 10 normal cerebellar tissues. Results: the abnormal expression rates of beta-catenin and VEGFR-2 in medulloblastoma were significantly higher than that in normal tissue. While the positive expression of SUFU gene in medulloblastoma was significantly lower than that in 10 normal cerebellar tissues. A significant negative correlation was found between beta-catenin and SUFU proteins and a positive correlation between beta-catenin and VEGFR-2 was found in medulloblastoma. Conclusion: Beta-catenin, VEGFR-2 and SUFU have important effects on the pathogenesis and development of medulloblastoma.

  15. Influenza A Virus NS1 Protein Promotes Efficient Nuclear Export of Unspliced Viral M1 mRNA.

    Science.gov (United States)

    Pereira, Carina F; Read, Eliot K C; Wise, Helen M; Amorim, Maria J; Digard, Paul

    2017-08-01

    Influenza A virus mRNAs are transcribed by the viral RNA-dependent RNA polymerase in the cell nucleus before being exported to the cytoplasm for translation. Segment 7 produces two major transcripts: an unspliced mRNA that encodes the M1 matrix protein and a spliced transcript that encodes the M2 ion channel. Export of both mRNAs is dependent on the cellular NXF1/TAP pathway, but it is unclear how they are recruited to the export machinery or how the intron-containing but unspliced M1 mRNA bypasses the normal quality-control checkpoints. Using fluorescent in situ hybridization to monitor segment 7 mRNA localization, we found that cytoplasmic accumulation of unspliced M1 mRNA was inefficient in the absence of NS1, both in the context of segment 7 RNPs reconstituted by plasmid transfection and in mutant virus-infected cells. This effect was independent of any major effect on steady-state levels of segment 7 mRNA or splicing but corresponded to a ∼5-fold reduction in the accumulation of M1. A similar defect in intronless hemagglutinin (HA) mRNA nuclear export was seen with an NS1 mutant virus. Efficient export of M1 mRNA required both an intact NS1 RNA-binding domain and effector domain. Furthermore, while wild-type NS1 interacted with cellular NXF1 and also increased the interaction of segment 7 mRNA with NXF1, mutant NS1 polypeptides unable to promote mRNA export did neither. Thus, we propose that NS1 facilitates late viral gene expression by acting as an adaptor between viral mRNAs and the cellular nuclear export machinery to promote their nuclear export.IMPORTANCE Influenza A virus is a major pathogen of a wide variety of mammalian and avian species that threatens public health and food security. A fuller understanding of the virus life cycle is important to aid control strategies. The virus has a small genome that encodes relatively few proteins that are often multifunctional. Here, we characterize a new function for the NS1 protein, showing that, as well as

  16. CED-4 is an mRNA-binding protein that delivers ced-3 mRNA to ribosomes.

    Science.gov (United States)

    Wang, Miao-xing; Itoh, Masanori; Li, Shimo; Hida, Yoko; Ohta, Kazunori; Hayakawa, Miki; Nishida, Emika; Ueda, Masashi; Islam, Saiful; Tana; Nakagawa, Toshiyuki

    2016-01-29

    Cell death abnormal (ced)-3 and ced-4 genes regulate apoptosis to maintain tissue homeostasis in Caenorhabditis elegans. Apoptosome formation and CED-4 translocation drive CED-3 activation. However, the precise role of CED-4 translocation is not yet fully understood. In this study, using a combination of immunoprecipitation and reverse transcription-polymerase chain reaction methods in cells and a glutathione-S-transferase pull down assay in a cell-free system, we show that CED-4 binds ced-3 mRNA. In the presence of ced-3 mRNA, CED-4 protein is enriched in the microsomal fraction and interacts with ribosomal protein L10a in mammalian cells, increasing the levels of CED-3. These results suggest that CED-4 forms a complex with ced-3 mRNA and delivers it to ribosomes for translation.

  17. Impaired gene expression of beta 1-adrenergic receptor, but not stimulatory G-protein Gs alpha, in rat ventricular myocardium treated with isoproterenol.

    Science.gov (United States)

    Kizaki, Keiichiro; Momozaki, Masami; Akatsuka, Keiko; Fujimori, Yuuki; Uchide, Tsuyoshi; Temma, Kyosuke; Hara, Yukio

    2004-07-01

    We investigated the gene expression of beta(1)-adrenergic receptor (beta(1)AR) and stimulatory G-protein Gsalpha, important signal transduction elements for regulating heart rate and contractility, in ventricle after chronic treatment with isoproterenol (ISO) in rat. Rats were treated with ISO (4 mg/kg, intraperitoneal) twice a day for 4 d. Ventricle weight of the heart and ventricle weight/body weight ratio were increased by 23% and 25% compared with control, respectively. Positive inotropic responses to ISO in left atrial muscle preparations isolated from ISO-treated rats were markedly decreased. Northern blot hybridization showed that the mRNA transcript of beta(1)AR was significantly decreased in ventricle of ISO-treated rats, whereas Gsalpha mRNA level was unchanged. Present results demonstrate that the gene expression of myocardial beta(1)AR, but not Gsalpha, was decreased in rat myocardium of ISO-induced cardiac hypertrophy, and suggesting that decrease in the gene expression of beta(1)AR may be one of the mechanisms responsible for the diminished cardiac function.

  18. Estrogen regulates the development of brain-derived neurotrophic factor mRNA and protein in the rat hippocampus.

    Science.gov (United States)

    Solum, Derek T; Handa, Robert J

    2002-04-01

    During development, estrogen has a variety of effects on morphological and electrophysiological properties of hippocampal neurons. Brain-derived neurotrophic factor (BDNF) also plays an important role in the survival and differentiation of neurons during development. We examined the effects of gonadectomy with and without estrogen replacement on the mRNA and protein of BDNF and its receptor, trkB, during early postnatal development of the rat hippocampus. We used immunocytochemistry to demonstrate that estrogen receptor alpha (ERalpha) and BDNF were localized to the same cells within the developing hippocampus. BDNF and ERalpha were colocalized in pyramidal cells of the CA3 subregion and to a lesser extent in CA1. To determine whether BDNF mRNA was regulated by estrogen during development, we gonadectomized male rat pups at postnatal day 0 (P0) and examined mRNA and protein levels from P0 to P25 using real-time reverse transcription-PCR and Western blot analysis. After gonadectomy, BDNF mRNA levels are significantly reduced on P7, but after treatment of gonadectomized animals with estradiol benzoate on P0, levels at all ages were similar to those in intact animals. BDNF mRNA changes after gonadectomy are accompanied by an increase in the levels of BDNF protein, which were reduced by estrogen treatment at P0. We also examined the effect of postnatal estrogen treatment on trkB. There were no significant changes in trkB mRNA or protein in gonadectomized or estrogen-replaced animals. These results suggest that a direct interaction may exist between ERalpha and BDNF to alter hippocampal physiology during development in the rat.

  19. The DEAD-Box Protein Dhh1p Couples mRNA Decay and Translation by Monitoring Codon Optimality.

    Science.gov (United States)

    Radhakrishnan, Aditya; Chen, Ying-Hsin; Martin, Sophie; Alhusaini, Najwa; Green, Rachel; Coller, Jeff

    2016-09-22

    A major determinant of mRNA half-life is the codon-dependent rate of translational elongation. How the processes of translational elongation and mRNA decay communicate is unclear. Here, we establish that the DEAD-box protein Dhh1p is a sensor of codon optimality that targets an mRNA for decay. First, we find mRNAs whose translation elongation rate is slowed by inclusion of non-optimal codons are specifically degraded in a Dhh1p-dependent manner. Biochemical experiments show Dhh1p is preferentially associated with mRNAs with suboptimal codon choice. We find these effects on mRNA decay are sensitive to the number of slow-moving ribosomes on an mRNA. Moreover, we find Dhh1p overexpression leads to the accumulation of ribosomes specifically on mRNAs (and even codons) of low codon optimality. Lastly, Dhh1p physically interacts with ribosomes in vivo. Together, these data argue that Dhh1p is a sensor for ribosome speed, targeting an mRNA for repression and subsequent decay.

  20. Rapid PCR-mediated synthesis of competitor molecules for accurate quantification of beta(2) GABA(A) receptor subunit mRNA.

    Science.gov (United States)

    Vela, J; Vitorica, J; Ruano, D

    2001-12-01

    We describe a fast and easy method for the synthesis of competitor molecules based on non-specific conditions of PCR. RT-competitive PCR is a sensitive technique that allows quantification of very low quantities of mRNA molecules in small tissue samples. This technique is based on the competition established between the native and standard templates for nucleotides, primers or other factors during PCR. Thus, the most critical parameter is the use of good internal standards to generate a standard curve from which the amount of native sequences can be properly estimated. At the present time different types of internal standards and methods for their synthesis have been described. Normally, most of these methods are time-consuming and require the use of different sets of primers, different rounds of PCR or specific modifications, such as site-directed mutagenesis, that need subsequent analysis of the PCR products. Using our method, we obtained in a single round of PCR and with the same primer pair, competitor molecules that were successfully used in RT-competitive PCR experiments. The principal advantage of this method is high versatility and economy. Theoretically it is possible to synthesize a specific competitor molecule for each primer pair used. Finally, using this method we have been able to quantify the increase in the expression of the beta(2) GABA(A) receptor subunit mRNA that occurs during rat hippocampus development.

  1. Heterologous Expression of Membrane and Soluble Proteins Derepresses GCN4 mRNA Translation in the Yeast Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Steffensen, L.; Pedersen, P. A.

    2006-01-01

    of the membrane-bound 1ß1 Na,K-ATPase from pig kidney, the rat pituitary adenylate cyclase seven-transmembrane-domain receptor, or a 401-residue soluble part of the Na,K-ATPase 1 subunit derepressed GCN4 mRNA translation up to 70-fold. GCN4 translation was very sensitive to the presence of heterologous protein......This paper describes the first physiological response at the translational level towards heterologous protein production in Saccharomyces cerevisiae. In yeast, the phosphorylation of eukaryotic initiation factor 2 (eIF-2 ) by Gcn2p protein kinase mediates derepression of GCN4 mRNA translation. Gcn4......, as a density of 1 of heterologous membrane protein derepressed translation maximally. Translational derepression of GCN4 was not triggered by misfolding in the endoplasmic reticulum, as expression of the wild type or temperature-sensitive folding mutants of the Na,K-ATPase increased GCN4 translation...

  2. Exercise training and work task induced metabolic and stress-related mRNA and protein responses in myalgic muscles

    DEFF Research Database (Denmark)

    Sjøgaard, Gisela; Zebis, Mette Kreutzfeldt; Kiilerich, Kristian;

    2013-01-01

    The aim was to assess mRNA and/or protein levels of heat shock proteins, cytokines, growth regulating, and metabolic proteins in myalgic muscle at rest and in response to work tasks and prolonged exercise training. A randomized controlled trial included 28 females with trapezius myalgia and 16...... healthy controls. Those with myalgia performed similar to 7 hrs repetitive stressful work and were subsequently randomized to 10 weeks of specific strength training, general fitness training, or reference intervention. Muscles biopsies were taken from the trapezius muscle at baseline, after work and after...... 10 weeks intervention. The main findings are that the capacity of carbohydrate oxidation was reduced in myalgic compared with healthy muscle. Repetitive stressful work increased mRNA content for heat shock proteins and decreased levels of key regulators for growth and oxidative metabolism...

  3. Exercise training and work task induced metabolic and stress-related mRNA and protein responses in myalgic muscles

    DEFF Research Database (Denmark)

    Sjøgaard, Gisela; Zebis, Mette Kreutzfeldt; Kiilerich, Kristian

    2013-01-01

    healthy controls. Those with myalgia performed similar to 7 hrs repetitive stressful work and were subsequently randomized to 10 weeks of specific strength training, general fitness training, or reference intervention. Muscles biopsies were taken from the trapezius muscle at baseline, after work and after...... 10 weeks intervention. The main findings are that the capacity of carbohydrate oxidation was reduced in myalgic compared with healthy muscle. Repetitive stressful work increased mRNA content for heat shock proteins and decreased levels of key regulators for growth and oxidative metabolism......The aim was to assess mRNA and/or protein levels of heat shock proteins, cytokines, growth regulating, and metabolic proteins in myalgic muscle at rest and in response to work tasks and prolonged exercise training. A randomized controlled trial included 28 females with trapezius myalgia and 16...

  4. Effect of the increased stability of the penicillin amidase mRNA on the protein expression levels.

    Science.gov (United States)

    Viegas, Sandra C; Schmidt, Dorothea; Kasche, Volker; Arraiano, Cecília M; Ignatova, Zoya

    2005-09-12

    Several factors at transcriptional, post-transcriptional or post-translational level determine the fate of a target protein and can severely restrict its yield. Here, we focus on the post-transcriptional regulation of the biosynthesis of the periplasmic protein, penicillin amidase (PA). The PA mRNA stability was determined under depleted RNase conditions in strains carrying single or multiple RNase deletions. Single deletion of the endonuclease RNase E yielded, as the highest, a fourfold stabilization of the PA mRNA. This effect, however, was reduced twice at post-translational level. The RNase II, generating secondary exonucleolytic cleavages in the mRNA, although not significantly influencing the PA mRNA decay, led also to an increase of the amount of mature PA. The non-proportional correlation between increased mRNA longevity and amount of active enzyme propose that the rational strategies for yield improvement must be based on a simultaneous tuning of more than one yield restricting factor.

  5. Small heat shock proteins inhibit amyloid-beta protein aggregation and cerebrovascular amyloid-beta protein toxicity.

    NARCIS (Netherlands)

    Wilhelmus, M.M.; Boelens, W.C.; Otte-Holler, I.; Kamps, B.; Waal, R.M.W. de; Verbeek, M.M.

    2006-01-01

    Small heat shock proteins Hsp20 and HspB2/B3 co-localize with Abeta deposition in senile plaques and cerebral amyloid angiopathy in Alzheimer's disease brains, respectively. It was the aim of our study to investigate if these and other sHsps bind to wild-type Abeta1-42 or the more toxic Abeta1-40

  6. Inflammatory Cytokines Stimulate Bone Morphogenetic Protein-2 Expression and Release from Pancreatic Beta Cells

    DEFF Research Database (Denmark)

    Urizar, Adriana Ibarra; Friberg, Josefine; Christensen, Dan Ploug;

    2016-01-01

    The proinflammatory cytokines interleukin-1 beta (IL-1β) and interferon gamma (IFN-γ) play important roles in the progressive loss of beta-cell mass and function during development of both type 1 and type 2 diabetes. We have recently showed that bone morphogenetic protein (BMP)-2 and -4...

  7. Validation of soluble amyloid-beta precursor protein assays as diagnostic CSF biomarkers for neurodegenerative diseases

    NARCIS (Netherlands)

    Waalwijk van Doorn, L.L.C. van; Koel-Simmelink, M.J.; Haussmann, U.; Klafki, H.; Struyfs, H.; Linning, P.; Knolker, H.J.; Twaalfhoven, H.; Kuiperij, H.B.; Engelborghs, S.; Scheltens, P.; Verbeek, M.M.; Vanmechelen, E.; Wiltfang, J.; Teunissen, C.E.

    2016-01-01

    Analytical validation of a biomarker assay is essential before implementation in clinical practice can occur. In this study, we analytically validated the performance of assays detecting soluble amyloid-beta precursor protein (sAPP) alpha and beta in CSF in two laboratories according to previously

  8. Is there a relationship between serum S-100 beta protein and neuropsychologic dysfunction after cardiopulmonary bypass?

    NARCIS (Netherlands)

    Westaby, S; Saatvedt, K; White, S; Katsumata, T; van Oeveren, W; Bhatnagar, NK; Brown, S; Halligan, PW

    2000-01-01

    Objectives: Over the past decade, the glial protein S-100 beta has been used to detect cerebral injury in a number of clinical settings including cardiac surgery. Previous investigations suggest that S-100 beta is capable of identifying patients with cerebral dysfunction after cardiopulmonary bypass

  9. Do post-translational beta cell protein modifications trigger type 1 diabetes?

    DEFF Research Database (Denmark)

    Størling, Joachim; Overgaard, Anne Julie; Brorsson, Caroline Anna;

    2013-01-01

    forms capable of specifically triggering beta cell destruction. In other immune-mediated diseases, autoantigens targeted by the immune system have undergone post-translational modification (PTM), thereby creating tissue-specific neo-epitopes. In a similar manner, PTM of beta cell proteins might create...

  10. Crystal structure of the human beta2 adrenergic G-protein-coupled receptor

    DEFF Research Database (Denmark)

    Rasmussen, Søren Gøgsig Faarup; Choi, Hee-Jung; Rosenbaum, Daniel M;

    2007-01-01

    Structural analysis of G-protein-coupled receptors (GPCRs) for hormones and neurotransmitters has been hindered by their low natural abundance, inherent structural flexibility, and instability in detergent solutions. Here we report a structure of the human beta2 adrenoceptor (beta2AR), which...

  11. Identifying mRNA, microRNA and protein profiles of melanoma exosomes.

    Directory of Open Access Journals (Sweden)

    Deyi Xiao

    Full Text Available BACKGROUND: Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and in various disease conditions. Tumor exosomes contain intact and functional mRNAs, small RNAs (including miRNAs, and proteins that can alter the cellular environment to favor tumor growth. Molecular profiling may increase our understanding of the role of exosomes in melanoma progression and may lead to discovery of useful biomarkers. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we used mRNA array profiling to identify thousands of exosomal mRNAs associated with melanoma progression and metastasis. Similarly, miRNA array profiling identified specific miRNAs, such as hsa-miR-31, -185, and -34b, involved in melanoma invasion. We also used proteomic analysis and discovered differentially expressed melanoma exosomal proteins, including HAPLN1, GRP78, syntenin-1, annexin A1, and annexin A2. Importantly, normal melanocytes acquired invasion ability through molecules transported in melanoma cell-derived exosomes. CONCLUSIONS/SIGNIFICANCE: Our results indicate that melanoma-derived exosomes have unique gene expression signatures, miRNA and proteomics profiles compared to exosomes from normal melanocytes. To the best of our knowledge, this is the first in-depth screening of the whole transcriptome/miRNome/proteome expression in melanoma exosomes. These results provide a starting point for future more in-depth studies of tumor-derived melanoma exosomes, which will aid our understanding of melanoma biogenesis and new drug-targets that may be translated into clinical applications, or as non-invasive biomarkers for melanoma.

  12. TP53 Promoter Methylation in Primary Glioblastoma: Relationship with TP53 mRNA and Protein Expression and Mutation Status

    OpenAIRE

    JESIONEK-KUPNICKA, DOROTA; Szybka, Malgorzata; Malachowska, Beata; Fendler, Wojciech; Potemski, Piotr; Piaskowski, Sylwester; Jaskolski, Dariusz; Papierz, Wielislaw; Skowronski, Wieslaw; Och, Waldemar; Kordek, Radzislaw; ZAWLIK, IZABELA

    2014-01-01

    Reduced expression of TP53 by promoter methylation has been reported in several neoplasms. It remains unclear whether TP53 promoter methylation is associated with reduced transcriptional and protein expression in glioblastoma (GB). The aim of our work was to study the impact of TP53 methylation and mutations on TP53 mRNA level and protein expression in 42 molecularly characterized primary GB tumors. We also evaluate the impact of all molecular alterations on the overall patient survival. The ...

  13. Absolute Quantification of Protein and mRNA Abundances Demonstrate Variability in GeneSpecific Translation Efficiency in Yeast

    DEFF Research Database (Denmark)

    Lahtvee, Petri-Jaan; Sanchez, Benjamin J.; Smialowska, Agata

    2017-01-01

    to model translation efficiencies and found that they vary more than 400-fold between genes. Non-linear regression analysis detected that mRNA abundance and translation elongation were the dominant factors controlling protein synthesis, explaining 61% and 15% of its variance. Metabolic flux balance......Protein synthesis is the most energy-consuming process in a proliferating cell, and understanding what controls protein abundances represents a key question in biology and biotechnology. We quantified absolute abundances of 5,354 mRNAs and 2,198 proteins in Saccharomyces cerevisiae under ten...

  14. The interactions between rat-adipose-derived stromal cells, recombinant human bone morphogenetic protein-2, and beta-tricalcium phosphate play an important role in bone tissue engineering.

    Science.gov (United States)

    E, Ling-Ling; Xu, Lu-Lu; Wu, Xia; Wang, Dong-Sheng; Lv, Yan; Wang, Jia-Zhu; Liu, Hong-Chen

    2010-09-01

    Cells, scaffolds, and growth factors are the three main factors for creating a stem-cell-based tissue-engineered construct, but the interactions between three factors are not very clear. We hereby explored the interactions between rat-adipose-derived stromal cells (rASCs), recombinant human bone morphogenetic protein-2 (rhBMP-2), and beta-tricalcium phosphate (beta-TCP) to provide evidence for their application in bone tissue engineering by evaluating the protein adsorption of beta-TCP, the cell attachment, alkaline phosphatase (ALP) activity/protein, osteocalcin (OCN) content, mineral formation, calcium content, phosphonium content, cell vitality, gene expression, and implantation in the backs of severe combined immunodeficient mice of rhBMP-2 preinducing rASCs seeded onto beta-TCP. The results showed that beta-TCP could adsorb the proteins from the media. The attachment, proliferation, and osteogenic properties of rASCs were supported by beta-TCP, as revealed using scanning electron microscopy. Compared with rASCs cultured on the culture plate, rASCs cultured on beta-TCP had significantly higher ALP activity/protein, OCN content, and mineral formation. These values for rASCs cultured on beta-TCP with rhBMP-2 increased most significantly. The rhBMP-2 significantly increased the calcium content, phosphonium content, and ALP, type I collagen, and OCN mRNA levels of rASCs cultured on beta-TCP. The methylthiazol tetrazolium method revealed that the vitality of rASCs cultured on beta-TCP with or without rhBMP-2 for 4, 7, and 28 days in vitro was insignificantly different. After 8 and 12 weeks of implantation, each group displayed increased bone formation over the 12-week period. The percentage of the new bone formed areas for beta-TCP/rhBMP-2 and beta-TCP was not significantly different. This value for rASCs/beta-TCP construct was significantly higher than that for beta-TCP group, but the maximal and robust bone formation was presented in rASCs/beta-TCP with rhBMP-2

  15. Interactions of protein kinase CK2beta subunit within the holoenzyme and with other proteins

    DEFF Research Database (Denmark)

    Kusk, M; Ahmed, R; Thomsen, B;

    1999-01-01

    in alpha-beta and beta-beta interactions. We also detected an intramolecular beta interaction within the amino acid stretch 132-165. Using CK2beta as a bait in a two-hybrid library screening several new putative cellular partners have been identified, among them the S6 kinase p90rsk, the putative tumor...

  16. Structural Basis for the Function of the Saccharomyces cerevisiae Gfd1 Protein in mRNA Nuclear Export* ♦

    OpenAIRE

    Zheng, Chao; Fasken, Milo B.; Marshall, Neil J.; Brockmann, Christoph; Rubinson, Max E.; Wente, Susan R.; Corbett, Anita H.; Stewart, Murray

    2010-01-01

    Following transcription, mRNA is processed, packaged into messenger ribonucleoprotein (mRNP) particles, and transported through nuclear pores (NPCs) to the cytoplasm. At the NPC cytoplasmic face, Dbp5 mediates mRNP remodeling and mRNA export factor dissociation, releasing transcripts for translation. In Saccharomyces cerevisiae, the conserved poly(A) RNA-binding protein, Nab2, facilitates NPC targeting of transcripts and also modulates poly(A) tail length. Dbp5 removes Nab2 from mRNPs at the ...

  17. Gestational Protein Restriction Increases Cardiac Connexin 43 mRNA levels in male adult rat offspring.

    Science.gov (United States)

    Rossini, Kamila Fernanda; Oliveira, Camila Andrea de; Rebelato, Hércules Jonas; Esquisatto, Marcelo Augusto Marreto; Catisti, Rosana

    2017-07-01

    The dietary limitation during pregnancy influences the growth and development of the fetus and offspring and their health into adult life. The mechanisms underlying the adverse effects of gestational protein restriction (GPR) in the development of the offspring hearts are not well understood. The aim of this study was to evaluate the effects of GPR on cardiac structure in male rat offspring at day 60 after birth (d60). Pregnant Wistar rats were fed a normal-protein (NP, 17% casein) or low-protein (LP, 6% casein) diet. Blood pressure (BP) values from 60-day-old male offspring were measured by an indirect tail-cuff method using an electro sphygmomanometer. Hearts (d60) were collected for assessment of connexin 43 (Cx43) mRNA expression and morphological and morphometric analysis. LP offspring showed no difference in body weight, although they were born lighter than NP offspring. BP levels were significantly higher in the LP group. We observed a significant increase in the area occupied by collagen fibers, a decrease in the number of cardiomyocytes by 104 µm2, and an increase in cardiomyocyte area associated with an increased Cx43 expression. GPR changes myocardial levels of Cx43 mRNA in male young adult rats, suggesting that this mechanism aims to compensate the fibrotic process by the accumulation of collagen fibers in the heart interstitium. A limitação dietética durante a gravidez influencia o crescimento e desenvolvimento do feto e da prole e sua saúde na vida adulta. Os mecanismos subjacentes dos efeitos adversos da restrição proteica gestacional (RPG) no desenvolvimento dos corações da prole não são bem compreendidos. Avaliar os efeitos da RPG sobre a estrutura cardíaca em filhotes machos de ratas aos 60 dias após o nascimento (d60). Ratos fêmeas Wistar grávidas foram alimentadas com uma dieta de proteína normal (PN, 17% caseína) ou de baixa proteína (BP, caseína 6%). Os valores de pressão arterial (PA) de descendentes do sexo masculino de

  18. Differential between Protein and mRNA Expression of CCR7 and SSTR5 Receptors in Crohn's Disease Patients

    Directory of Open Access Journals (Sweden)

    Nathalie Taquet

    2009-01-01

    Full Text Available Crohn's disease (CD is a multifactorial chronic inflammatory bowel disease of unknown cause. The aim of the present study was to explore if mRNA over-expression of SSTR5 and CCR7 found in CD patients could be correlated to respective protein expression. When compared to healthy donors, SSTR5 was over-expressed 417 ± 71 times in CD peripheral blood mononuclear cells (PBMCs. Flow cytometry experiments showed no correlation between mRNA and protein expression for SSTR5 in PBMCs. In an attempt to find a reason of such a high mRNA expression, SSTR5 present on CD PBMCs were tested and found as biologically active as on healthy cells. In biopsies of CD intestinal tissue, SSTR5 was not over-expressed but CCR7, unchanged in PBMCs, was over-expressed by 10 ± 3 times in the lamina propria. Confocal microscopy showed a good correlation of CCR7 mRNA and protein expression in CD intestinal biopsies. Our data emphasize flow and image cytometry as impossible to circumvent in complement to molecular biology so to avoid false interpretation on receptor expressions. Once confirmed by further large-scale studies, our preliminary results suggest a role for SSTR5 and CCR7 in CD pathogenesis.

  19. A network of PUF proteins and Ras signaling promote mRNA repression and oogenesis in C. elegans.

    Science.gov (United States)

    Hubstenberger, Arnaud; Cameron, Cristiana; Shtofman, Rebecca; Gutman, Shiri; Evans, Thomas C

    2012-06-15

    Cell differentiation requires integration of gene expression controls with dynamic changes in cell morphology, function, and control. Post-transcriptional mRNA regulation and signaling systems are important to this process but their mechanisms and connections are unclear. During C. elegans oogenesis, we find that two groups of PUF RNA binding proteins (RNABPs), PUF-3/11 and PUF-5/6/7, control different specific aspects of oocyte formation. PUF-3/11 limits oocyte growth, while PUF-5/6/7 promotes oocyte organization and formation. These two PUF groups repress mRNA translation through overlapping but distinct sets of 3' untranslated regions (3'UTRs). Several PUF-dependent mRNAs encode other mRNA regulators suggesting both PUF groups control developmental patterning of mRNA regulation circuits. Furthermore, we find that the Ras-MapKinase/ERK pathway functions with PUF-5/6/7 to repress specific mRNAs and control oocyte organization and growth. These results suggest that diversification of PUF proteins and their integration with Ras-MAPK signaling modulates oocyte differentiation. Together with other studies, these findings suggest positive and negative interactions between the Ras-MAPK system and PUF RNA-binding proteins likely occur at multiple levels. Changes in these interactions over time can influence spatiotemporal patterning of tissue development.

  20. Aldosterone and thyroid hormone modulation of alpha 1-, beta 1-mRNA, and Na,K-pump sites in rabbit distal colon epithelium. Evidence for a novel mechanism of escape from the effect of hyperaldosteronemia.

    Science.gov (United States)

    Wiener, H; Nielsen, J M; Klaerke, D A; Jørgensen, P L

    1993-05-01

    Aldosterone and thyroid hormone regulation of Na,K-pump biosynthesis has been examined in the distal colon epithelium of rabbits. Qualitative analysis of alpha-subunit isoform distribution (alpha 1, alpha 2, alpha 3) detected only the alpha 1-mRNA in the distal colon epithelium and outer renal medulla, while all three isoforms were observed in rabbit brain. A low-sodium diet led to a rise in serum aldosterone from 0.6 nM to 1.4-1.9 nM and an increase of alpha 1-mRNA to 162%, beta 1-mRNA to 120%, and the number of Na,K-pump units as determined by specific [3H]-ouabain binding to 182% of control by the second day of the diet. While aldosterone levels remained elevated, a spontaneous decrease in serum levels of T3 and T4 to 50-60% of control from the third day of the diet was followed by downregulation of beta 1-mRNA to 55-67%, alpha 1-mRNA to 63-105%, and of [3H]-ouabain binding to 103% of control, suggesting that a reduced rate of synthesis of the beta 1-subunit is rate limiting for Na,K-pump biosynthesis. Substitution with T3 (10 micrograms/kg) at the seventh day with transient restoration of serum T3 to control levels, led to rapid accumulation of beta 1-mRNA to 152%, of alpha 1-mRNA to 135%, and of the number of Na,K-pump units to 153% of control. This is consistent with thyroid hormone having a permissive role for the aldosterone stimulation of Na,K-pump biosynthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Quantitative detection of metastasis-associated1mRNA and protein expression in breast cancer%Quantitative detection of metastasis-associated 1 mRNA and protein expression in breast cancer

    Institute of Scientific and Technical Information of China (English)

    Hiroko Yamashita; Tatsuya Toyama; Hiroshi Sugiura; Mei ZHANG; Shunzo Kobayashi; Yoshitaka Fujii; Hirotaka Iwase; Zhenhuan ZHANG

    2013-01-01

    Objective Understanding the mechanism of breast cancer metastasis will benefit those patients in need of aggressive treatment and avoid side effects caused by chemotherapy over treatment.Recently,a potential metastasis-associated gene and its product,the metastasis-associated 1 (MTA1),were identified; this gene has been found to be overexpressed in a variety of cancers.Methods In the present study,therefore,the level of expression of MTA1 mRNA has been assessed by LightCycler quantitative real-time RT-PCR in 160 cases of invasive carcinoma of the breast.MTA1 protein expression level was also detected by immunohistochemistry from available paired tissues of 154 cases.Associations between MTA1 mRNA and protein expression and clinicopathological factors were analyzed.Results It was found that MTA1 mRNA was expressed at significantly higher levels in patients with negative lymph node status,with ER and PgR positive and HER2 negative tumor.No difference was found between patient age,tumor size and histological grade groups.Patients with high levels of expression of MTA1 mRNA had a better prognosis than those with low expression.However,no difference was found between the protein level and clinicopathological factors.Univariate and multivariate prognostic analysis did not demonstrate that MTA1 mRNA was an independent prognostic factor for breast cancer.Conclusion In breast cancer,inconsistent with other tumor types,MTA1 gene expression is correlated with non-invasive clinicopathological factors and longer survival,which might suggest MTA1 gene is a tumor type specific metastasis associated gene.

  2. Overexpression of protease nexin-1 mRNA and protein in oral squamous cell carcinomas

    DEFF Research Database (Denmark)

    Gao, Shan; Krogdahl, Annelise; Sørensen, Jens Ahm;

    2007-01-01

    -1 has been almost totally neglected. We have now compared the level of PN-1 mRNA in 20 cases of oral squamous cell carcinomas and in matched samples of the corresponding normal oral tissues. We found that the average PN-1 mRNA level in tumours and normal tissues was significantly different, being...... increased up to 13 fold in tumour samples compared with the average level in normal tissues. The PN-1 mRNA level was significantly higher in tumours from patients with lymph node metastasis than in tumours from patients without. We could conclude that PN-1 is frequently overexpressed in oral squamous cell...

  3. Induction of hepatic carbonyl reductase/20{beta}-hydroxysteroid dehydrogenase mRNA in rainbow trout downstream from sewage treatment works-Possible roles of aryl hydrocarbon receptor agonists and oxidative stress

    Energy Technology Data Exchange (ETDEWEB)

    Albertsson, E., E-mail: eva.albertsson@zool.gu.se [Department of Zoology, University of Gothenburg, Box 463, SE-405 30 Goeteborg (Sweden); Larsson, D.G.J. [Department of Neuroscience and Physiology, Sahlgrenska Academy at the University of Gothenburg, Box 434, SE-405 30 Goeteborg (Sweden); Foerlin, L. [Department of Zoology, University of Gothenburg, Box 463, SE-405 30 Goeteborg (Sweden)

    2010-05-05

    Carbonyl reductase/20{beta}-hydroxysteroid dehydrogenase (CR/20{beta}-HSD) serves both as a key enzyme in the gonadal synthesis of maturing-inducing hormone in salmonids, and as an enzyme protecting against certain reactive oxygen species. We have previously shown that mRNA of the hepatic CR/20{beta}-HSD B isoform is increased in rainbow trout caged downstream from a Swedish sewage treatment plant. Here, we report an increase of both the A as well as B form in fish kept downstream from a second sewage treatment plant. The two mRNAs were also induced in fish hepatoma cells in vitro after exposure to effluent extract. This indicates that the effects observed in vivo could be a direct effect on the liver, i.e. the mRNA induction does not require a signal from any other organ. When fish were exposed in vivo to several effluents treated with more advanced methods (ozone, moving bed biofilm reactor or membrane bioreactor) the expression of hepatic mRNA CR/20{beta}-HSD A and B was significantly reduced. Their abundance did not parallel the reduction of estrogen-responsive transcripts, in agreement with our previous observations that ethinylestradiol is not a potent inducer. Treatment with norethisterone, methyltestosterone or hydrocortisone in vivo did not induce the hepatic CR/20{beta}-HSD A and B mRNA expression. In contrast, both isoforms were markedly induced by the aryl hydrocarbon receptor agonist {beta}-naphthoflavone as well as by the pro-oxidant herbicide paraquat. We hypothesize that the induction of CR/20{beta}-HSD A and B by sewage effluents could be due to anthropogenic contaminants stimulating the aryl hydrocarbon receptor and/or causing oxidative stress.

  4. Immunolocalization of alpha-keratins and associated beta-proteins in lizard epidermis shows that acidic keratins mix with basic keratin-associated beta-proteins.

    Science.gov (United States)

    Alibardi, Lorenzo

    2014-07-01

    The differentiation of the corneous layers of lizard epidermis has been analyzed by ultrastructural immunocytochemistry using specific antibodies against alpha-keratins and keratin associated beta-proteins (KAbetaPs, formerly indicated as beta-keratins). Both beta-cells and alpha-cells of the corneous layer derive from the same germinal layer. An acidic type I alpha-keratin is present in basal and suprabasal layers, early differentiating clear, oberhautchen, and beta-cells. Type I keratin apparently disappears in differentiated beta- and alpha-layers of the mature corneous layers. Conversely, a basic type II alpha-keratin rich in glycine is absent or very scarce in basal and suprabasal layers and this keratin likely does not pair with type I keratin to form intermediate filaments but is weakly detected in the pre-corneous and corneous alpha-layer. Single and double labeling experiments show that in differentiating beta-cells, basic KAbetaPs are added and replace type-I keratin to form the hard beta-layer. Epidermal alpha-keratins contain scarce cysteine (0.2-1.4 %) that instead represents 4-19 % of amino acids present in KAbetaPs. Possible chemical bonds formed between alpha-keratins and KAbetaPs may derive from electrostatic interactions in addition to cross-linking through disulphide bonds. Both the high content in glycine of keratins and KAbetaPs may also contribute to increase the hydrophobicy of the beta- and alpha-layers and the resistance of the corneous layer. The increase of gly-rich KAbetaPs amount and the bonds to the framework of alpha-keratins give rise to the inflexible beta-layer while the cys-rich KAbetaPs produce a pliable alpha-layer.

  5. Proteomic analysis of ACTN4-interacting proteins reveals it's a putative involvement in mRNA metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Khotin, Mikhail, E-mail: h_mg@mail.ru [Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky av., 4, 194064 St. Petersburg (Russian Federation); Turoverova, Lidia [Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky av., 4, 194064 St. Petersburg (Russian Federation); Aksenova, Vasilisa [Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky av., 4, 194064 St. Petersburg (Russian Federation); Department of Genetics, St. Petersburg State University, Universitetskaya nab., 7/9, 199034 St. Petersburg (Russian Federation); Barlev, Nikolai [Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky av., 4, 194064 St. Petersburg (Russian Federation); Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN (United Kingdom); Borutinskaite, Veronika Viktorija [Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Linkoeping University, SE-581 85 Linkoeping (Sweden); Department of Developmental Biology, Institute of Biochemistry, LT-08662 Vilnius (Lithuania); Vener, Alexander [Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Linkoeping University, SE-581 85 Linkoeping (Sweden); Bajenova, Olga [Department of Genetics, St. Petersburg State University, Universitetskaya nab., 7/9, 199034 St. Petersburg (Russian Federation); Magnusson, Karl-Eric [Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Linkoeping University, SE-581 85 Linkoeping (Sweden); Pinaev, George P. [Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky av., 4, 194064 St. Petersburg (Russian Federation); Tentler, Dmitri, E-mail: dtentler@mail.cytspb.rssi.ru [Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky av., 4, 194064 St. Petersburg (Russian Federation)

    2010-06-25

    Alpha-actinin 4 (ACTN4) is an actin-binding protein. In the cytoplasm, ACTN4 participates in structural organisation of the cytoskeleton via cross-linking of actin filaments. Nuclear localisation of ACTN4 has also been reported, but no clear role in the nucleus has been established. In this report, we describe the identification of proteins associated with ACTN4 in the nucleus. A combination of two-dimensional gel electrophoresis (2D-GE) and MALDI-TOF mass-spectrometry revealed a large number of ACTN4-bound proteins that are involved in various aspects of mRNA processing and transport. The association of ACTN4 with different ribonucleoproteins suggests that a major function of nuclear ACTN4 may be regulation of mRNA metabolism and signaling.

  6. Expression of intestinal mucosa tight junctions claudin proteins and mRNA in patients with irritable bowel syndrome

    Institute of Scientific and Technical Information of China (English)

    KONG Wu-ming; GONG Jun; DONG Lei; LU Xiao-lan; XU Jun-rong

    2007-01-01

    Objective:To investigate the changes of intestinal mucosa tight junctions(TJs)claudin-1,-3,-4 proteins and mRNA changes in patients with irritable bowel syndrome(IBS)and to elucidate their possible roles in the changes of bowel evacuation habit and formation.Methods:Claudin-1,-3,-4 proteins and mRNA were evaluated in intestinal mucosa in control group,D-IBS(diarrhea IBS)group and C-IBS (constipation IBS)group with immunohistochemical assay and Realtime-PCR.Results:It was observed that claudin-1,-3,-4 proteins were localized in the membranes of epithelial cells along the entire length of the plasma membrane including the apical end of the epithelial cells.The claudins were concentrated at the site of TJs only.Claudin-1,-3,-4 mRNA and claudin-1 protein in small intestinal mucosa and colonal mucous in D-IBS group were significantly downregulated(P<0.05).Claudin-1,-3,-4 mRNA and proteins in small intestinal mucosa and co1onal mucous in C-IBS group were significantly upregulated(P<0.05).There was no significant difference in the expression of claudin-3 protein in both small intestinal mucosa and colonal mucous between D-IBS group and control group(P>0.05).Similarly,no significantly different expression of claudin-4 protein in colonal mucous in D-IBS group was found compared with control group(P>0.05).Otherwise,the expression of claudin-4 protein in small intestinal mucosa decreased in D-IBS group(P<0.05).Conclusion:Claudin-1,-3,-4 may play a potential important role in the changes of bowel evacuation habit and formation in patients with IBS.It is not due to the localization changes of claudin proteins in TJ,but may be caused by the quantitative changes of the expression of TJ proteins and mRNA.

  7. Cloning and characterization of DNA complementary to the canine distemper virus mRNA encoding matrix, phosphoprotein, and nucleocapsid protein

    Energy Technology Data Exchange (ETDEWEB)

    Rozenblatt, S.; Eizenberg, O.; Englund, G.; Bellini, W.J.

    1985-02-01

    Double-stranded cDNA synthesized from total polyadenylate-containing mRNA, extracted from monkey kidney cells infected with canine distemper virus (CDV), has been cloned into the PstI site of Escherichia coli plasmid pBR322. Clones containing canine distemper virus DNA were identified by hybridization to a canine distemper virus-specific, /sup 32/P-labeled cDNA. Four specific clones containing different classes of sequences have been identified. The cloned plasmids contain inserts of 800 (clone 44-80), 960 (clone 74-16), 1700 (clone 364), and 950 (clone 40-9) base pairs. The sizes of the mRNA species complementary to these inserts are 1500, 1850, 1850 and 2500 nucleotides, respectively, as determined by the Northern technique. Three of the cloned DNA fragments were further identified as the reverse transcripts of the mRNA coding for the matrix, phosphoprotein, and nucleocapsid protein of CDV.

  8. Exosome-bound WD repeat protein Monad inhibits breast cancer cell invasion by degrading amphiregulin mRNA.

    Directory of Open Access Journals (Sweden)

    Makio Saeki

    Full Text Available Increased stabilization of mRNA coding for key cancer genes can contribute to invasiveness. This is achieved by down-regulation of exosome cofactors, which bind to 3'-UTR in cancer-related genes. Here, we identified amphiregulin, an EGFR ligand, as a target of WD repeat protein Monad, a component of R2TP/prefoldin-like complex, in MDA-MB-231 breast cancer cells. Monad specifically interacted with both the 3'-UTR of amphiregulin mRNA and the RNA degrading exosome, and enhanced decay of amphiregulin transcripts. Knockdown of Monad increased invasion and this effect was abolished with anti-amphiregulin neutralizing antibody. These results suggest that Monad could prevent amphiregulin-mediated invasion by degrading amphiregulin mRNA.

  9. Exercise Training and Work Task Induced Metabolic and Stress-Related mRNA and Protein Responses in Myalgic Muscles

    Directory of Open Access Journals (Sweden)

    Gisela Sjøgaard

    2013-01-01

    Full Text Available The aim was to assess mRNA and/or protein levels of heat shock proteins, cytokines, growth regulating, and metabolic proteins in myalgic muscle at rest and in response to work tasks and prolonged exercise training. A randomized controlled trial included 28 females with trapezius myalgia and 16 healthy controls. Those with myalgia performed ~7 hrs repetitive stressful work and were subsequently randomized to 10 weeks of specific strength training, general fitness training, or reference intervention. Muscles biopsies were taken from the trapezius muscle at baseline, after work and after 10 weeks intervention. The main findings are that the capacity of carbohydrate oxidation was reduced in myalgic compared with healthy muscle. Repetitive stressful work increased mRNA content for heat shock proteins and decreased levels of key regulators for growth and oxidative metabolism. In contrast, prolonged general fitness as well as specific strength training decreased mRNA content of heat shock protein while the capacity of carbohydrate oxidation was increased only after specific strength training.

  10. Exercise training and work task induced metabolic and stress-related mRNA and protein responses in myalgic muscles.

    Science.gov (United States)

    Sjøgaard, Gisela; Zebis, Mette K; Kiilerich, Kristian; Saltin, Bengt; Pilegaard, Henriette

    2013-01-01

    The aim was to assess mRNA and/or protein levels of heat shock proteins, cytokines, growth regulating, and metabolic proteins in myalgic muscle at rest and in response to work tasks and prolonged exercise training. A randomized controlled trial included 28 females with trapezius myalgia and 16 healthy controls. Those with myalgia performed ~7 hrs repetitive stressful work and were subsequently randomized to 10 weeks of specific strength training, general fitness training, or reference intervention. Muscles biopsies were taken from the trapezius muscle at baseline, after work and after 10 weeks intervention. The main findings are that the capacity of carbohydrate oxidation was reduced in myalgic compared with healthy muscle. Repetitive stressful work increased mRNA content for heat shock proteins and decreased levels of key regulators for growth and oxidative metabolism. In contrast, prolonged general fitness as well as specific strength training decreased mRNA content of heat shock protein while the capacity of carbohydrate oxidation was increased only after specific strength training.

  11. Dynamics of glucose-induced membrane recruitment of protein kinase C beta II in living pancreatic islet beta-cells.

    Science.gov (United States)

    Pinton, Paolo; Tsuboi, Takashi; Ainscow, Edward K; Pozzan, Tullio; Rizzuto, Rosario; Rutter, Guy A

    2002-10-01

    The mechanisms by which glucose may affect protein kinase C (PKC) activity in the pancreatic islet beta-cell are presently unclear. By developing adenovirally expressed chimeras encoding fusion proteins between green fluorescent protein and conventional (betaII), novel (delta), or atypical (zeta) PKCs, we show that glucose selectively alters the subcellular localization of these enzymes dynamically in primary islet and MIN6 beta-cells. Examined by laser scanning confocal or total internal reflection fluorescence microscopy, elevated glucose concentrations induced oscillatory translocations of PKCbetaII to spatially confined regions of the plasma membrane. Suggesting that increases in free cytosolic Ca(2+) concentrations ([Ca(2+)](c)) were primarily responsible, prevention of [Ca(2+)](c) increases with EGTA or diazoxide completely eliminated membrane recruitment, whereas elevation of cytosolic [Ca(2+)](c) with KCl or tolbutamide was highly effective in redistributing PKCbetaII both to the plasma membrane and to the surface of dense core secretory vesicles. By contrast, the distribution of PKCdelta.EGFP, which binds diacylglycerol but not Ca(2+), was unaffected by glucose. Measurement of [Ca(2+)](c) immediately beneath the plasma membrane with a ratiometric "pericam," fused to synaptic vesicle-associated protein-25, revealed that depolarization induced significantly larger increases in [Ca(2+)](c) in this domain. These data demonstrate that nutrient stimulation of beta-cells causes spatially and temporally complex changes in the subcellular localization of PKCbetaII, possibly resulting from the generation of Ca(2+) microdomains. Localized changes in PKCbetaII activity may thus have a role in the spatial control of insulin exocytosis.

  12. Maintenance of Pdx1 mRNA translation in islet β-cells during the unfolded protein response.

    Science.gov (United States)

    Templin, Andrew T; Maier, Bernhard; Tersey, Sarah A; Hatanaka, Masayuki; Mirmira, Raghavendra G

    2014-11-01

    In type 1 diabetes, proinflammatory cytokines secreted by infiltrating immune cells activate the unfolded protein response (UPR) in islet β-cells, which leads to attenuation of global mRNA translation. Under such conditions, privileged mRNAs required for adaptation to the prevailing stress are maintained in an actively translated state. Pdx1 is a β-cell transcription factor that is required for the adaptive UPR, but it is not known how translation of its mRNA is maintained under these conditions. To study translation, we established conditions in vitro with MIN6 cells and mouse islets and a mixture of proinflammatory cytokines (IL-1β, TNF-α, and IFN-γ) that mimicked the UPR conditions seen in type 1 diabetes. Cell extracts were then subjected to polyribosome profiling to monitor changes to mRNA occupancy by ribosomes. Similar to other privileged mRNAs (Atf4 and Chop), Pdx1 mRNA remained partitioned in actively translating polyribosomes under the UPR, whereas the mRNA encoding a proinsulin-processing enzyme (Cpe) and others partitioned into inactively translating monoribosomes. Bicistronic luciferase reporter analyses revealed that the distal portion of the 5'-untranslated region of mouse Pdx1 (between bp -105 to -280) contained elements that promoted translation under both normal and UPR conditions, and this region exhibited conserved sequences and secondary structure similar to those of other known internal ribosome entry sites. Our findings suggest that Pdx1 protein levels are maintained in the setting of the UPR, in part, through elements in the 5'-untranslated region that confer privileged mRNA translation in a 5'-7-methylguanylate cap-independent manner.

  13. Superficial zone protein (lubricin) in the different tissue compartments of the knee joint: modulation by transforming growth factor beta 1 and interleukin-1 beta.

    Science.gov (United States)

    Lee, Sang Yang; Niikura, Takahiro; Reddi, A Hari

    2008-11-01

    Superficial-zone protein (SZP), also known as lubricin, is a key mediator of boundary lubrication and plays an important role in the functional integrity of the diarthrodial joint. The aim of this investigation was to examine the role of transforming growth factor beta (TGF-beta) and interleukin-1 beta (IL-1beta) on the expression of SZP in various compartments of the bovine knee joint: the superficial zone of articular cartilage, synovium, meniscus, and anterior and posterior cruciate ligaments. The effects of TGF-beta1 and IL-1beta on SZP expression were examined in explants and cells from the different tissue compartments. TGF-beta1 up-regulated the expression of SZP in cultured explants, but IL-1beta down-regulated it. Quantitative analysis of secreted proteins in the medium of the cells demonstrated significant stimulation by TGF-beta1 and inhibition by IL1-beta of the accumulation of SZP protein in all four tissues. Real-time polymerase chain reaction analysis revealed that TGF-beta1 significantly up-regulated SZP expression and that IL-1beta down-regulated it. These results revealed the modulation of SZP expression in various compartments of the knee joint by TGF-beta1 and IL-1beta. In addition, SZP was found to be immunolocalized at the surface layer of cells in histological sections of all four tissue compartments. Collectively, results of the current study on regulation of SZP expression by TGF-beta and IL-1 help provide new insights, into tissue engineering strategies to repair and regenerate the different tissue compartments in the articular joint with optimal lubrication.

  14. Interferon gamma, interleukin 4 and transforming growth factor beta in experimental autoimmune encephalomyelitis in Lewis rats: dynamics of cellular mRNA expression in the central nervous system and lymphoid cells

    DEFF Research Database (Denmark)

    Issazadeh-Navikas, Shohreh; Mustafa, M; Ljungdahl, A;

    1995-01-01

    The potential role of certain important immunoregulatory and effector cytokines in autoimmune neuroinflammation have been studied. We have examined the expression of mRNA, with in situ hybridization, of interferon gamma (IFN-gamma), interleukin 4 (IL-4) and transforming growth factor beta (TGF...

  15. First structural evidence of sequestration of mRNA cap structures by type 1 ribosome inactivating protein from Momordica balsamina.

    Science.gov (United States)

    Kushwaha, Gajraj Singh; Yamini, Shavait; Kumar, Mukesh; Sinha, Mau; Kaur, Punit; Sharma, Sujata; Singh, Tej P

    2013-05-01

    This is the first structural evidence of recognition of mRNA cap structures by a ribosome inactivating protein. It is well known that a unique cap structure is formed at the 5' end of mRNA for carrying out various processes including mRNA maturation, translation initiation, and RNA turnover. The binding studies and crystal structure determinations of type 1 ribosome inactivating protein (RIP-1) from Momordica balsamina (MbRIP-1) were carried out with mRNA cap structures including (i) N7-methyl guanine (m7G), (ii) N7-methyl guanosine diphosphate (m7GDP), and (iii) N7-methyl guanosine triphosphate (m7GTP). These compounds showed affinities to MbRIP-1 at nanomolar concentrations. The structure determinations of the complexes of MbRIP-1 with m7G, m7GDP, and m7GTP at 2.65, 1.77, and 1.75 Å resolutions revealed that all the three compounds bound to MbRIP-1 in the substrate binding site at the positions which are slightly shifted towards Glu85 as compared to those of rRNA substrates. In this position, Glu85 forms several hydrogen bonds with guanine moiety while N-7 methyl group forms van der Waals contacts. However, the guanine rings are poorly stacked in these complexes. Thus, the mode of binding by MbRIP-1 to mRNA cap structures is different which results in the inhibition of depurination. Since some viruses are known to exploit the capping property of the host, this action of MbRIP-1 may have implications for the antiviral activity of this protein in vivo. The understanding of the mode of binding of MbRIP-1 to cap structures may also assist in the design of anti-viral agents. Copyright © 2013 Wiley Periodicals, Inc.

  16. Cerebral microvascular amyloid beta protein deposition induces vascular degeneration and neuroinflammation in transgenic mice expressing human vasculotropic mutant amyloid beta precursor protein.

    NARCIS (Netherlands)

    Miao, J.; Xu, F.; Davis, J.; Otte-Holler, I.; Verbeek, M.M.; Nostrand, W.E. van

    2005-01-01

    Cerebral vascular amyloid beta-protein (Abeta) deposition, also known as cerebral amyloid angiopathy, is a common pathological feature of Alzheimer's disease. Additionally, several familial forms of cerebral amyloid angiopathy exist including the Dutch (E22Q) and Iowa (D23N) mutations of Abeta. Incr

  17. Porcine blood mononuclear cell cytokine responses to PAMP molecules: comparison of mRNA and protein production

    DEFF Research Database (Denmark)

    Sørensen, Nanna Skall; Skovgaard, Kerstin; Heegaard, Peter M. H.

    2011-01-01

    -α and IL-12 p40, and PGN, LPS and Pam3Cys inducing varying amounts of IL-12 p40, IL-1β, TNF-α, IL-6 and IL-10. Surprisingly, the ssRNA-mimic poly-U induced IL-6 and IL-1β only. Using CpG, PGN and LPS, the kinetics of cytokine production measured as mRNA (reverse transcription (RT)-qPCR) and protein (ELISA...... the induction of IFN-α, IL-12 p40, IL-1β, TNF-α, IL-6 and IL-10 by PAMP-molecules [CpG oligonucleotide D19 (CpG), peptidoglycan (PGN), lipopolysaccharide (LPS), Pam3Cys and poly-U] in porcine blood mononuclear cells (BMC) within a 24h period. As expected, cytokine responses were PAMP-specific, CpG inducing IFN......), respectively, correlated well, mRNA responses preceding protein responses. With the exception of IL-1β and IL-6, mRNA-responses were transient, whereas protein responses, except for TNF-α, followed saturation kinetics. Remarkably, LPS-induced TNF-α mRNA was not followed by a protein response. These results...

  18. EXPRESSION OF PEDF mRNA AND PROTEIN IN NORMAL MOUSE RETINA AND EXPERIMENTAL CHOROIDAL NEO-VASCULARIZATION TISSUES

    Institute of Scientific and Technical Information of China (English)

    张雷; 王康孙; 王玲; 张士胜; 石海云

    2004-01-01

    Objective To study the expression of pigment epithelium derived factor ( PEDF) in normal mouse retina and experimental choroidal neovascularization (CNV) tissues. Methods CNV mouse models were induced by diode laser. The expression of PEDF mRNA and protein in normal mouse retina and CNV tissues were detected by in situ hybridization and immunohistochemical study. Results In normal mouse retina,PEDF mRNA was observed in the ganglion cell layer, inner nuclear layer and RPE cell layer, and PEDF protein was observed mainly in the nerve fiber layer, ganglion cell layer, photoreceptor cell layer and RPE cell layer, and lower level expression of PEDF protein was also observed in the inner plexiform layer and outer plexiform layer. In CNV tissues,the expression of PEDF mRNA and protein was also observed. 3d and 1 week after photocoagulation, the expression level of PEDF was relatively lower, and increased following the development of CNV. The level was the highest 2 weeks after photocoagulation, then decreased at 3 weeks. Conclusion PEDF was expressed in different layers of retina and was obviously expressed in the CNV tissues induced by laser photocoagulation. These findings suggest that PEDF may participate and modulate the development of CNV.

  19. [Effect of dragon's blood on TGF-beta/smads signal transduction molecule mRNA expression in the lung tissue of rats with pulmonary fibrosis].

    Science.gov (United States)

    Nie, Li; Zheng, Bi-xia; Cheng, De-yun; Yang, Li-teng; Mu, Mao; Hu, Xiao-bo; Fang, Xun

    2007-09-01

    To investigate the effect of Dragon's Blood on the expression of TGF-beta signal transduction molecule TGFbetaR II or Smad4 mRNA in the lung tissue of rats with pulmonary fibrosis, and to evaluate the effect and its mechanism of Dragon's Blood on pulmonary fibrosis. 30 SD rats were randomly divided into three groups: fibrosis model, treatment and normal control groups. In model group and treatment group, the pulmonary fibrous tissues were induced to form with the intratracheal injection of bleomycin (5 mg/kg). In normal control group, saline was given intratracheally. Dragon's Blood was administered intragastricly in treatment group with a dose of 180 mg/kg diluted in 2 mL saline while saline was given intragastricly to other two groups with same volume from day 2 till day 28 after modeling. All rats were sacrificed on the 29th day. The rat lung histopathology was examined with HE staining. In situ hybridization was used to detect the expressions of TGFbetaR II and Smad4 mRNAs in lung tissue, and the expression of collagen fibril I was examined by an immunohistochemical staining. The inflammation cell counting in treatment group (12913.78 +/- 5640.12) was significant lower than that in model group (22243.60 +/- 5011.55, P 0.05). The expression of collagen fibril I in the lung tissue of rats in treatment group was significant lower than that in model group (P Dragon's Blood can effectively reduce rats' pulmonary fibrosis, of which the mechanisms may be to inhibit the expression of TGFbetaR II mRNA in the lung tissue and thus to have the preventive effect on the excessive deposit of collagen fibril I.

  20. Lentivectors encoding immunosuppressive proteins genetically engineer pancreatic beta-cells to correct diabetes in allogeneic mice.

    Science.gov (United States)

    Kojaoghlanian, T; Joseph, A; Follenzi, A; Zheng, J H; Leiser, M; Fleischer, N; Horwitz, M S; DiLorenzo, T P; Goldstein, H

    2009-03-01

    The effectiveness of genetic engineering with lentivectors to protect transplanted cells from allogeneic rejection was examined using, as a model, type 1 diabetes treatment with beta-cell transplantation, whose widespread use has been limited by the requirement for sustained immunosuppressive treatment to prevent graft rejection. We examined whether lentivectors expressing select immunosuppressive proteins encoded by the adenoviral genome early region 3 (AdE3) would protect transplanted beta-cells from an alloimmune attack. The insulin-producing beta-cell line beta TC-tet (C3HeB/FeJ-derived) was transduced with lentiviruses encoding the AdE3 proteins gp19K and RID alpha/beta. The efficiency of lentiviral transduction of beta TC-tet cells exceeded 85%. Lentivector expression of gp19K decreased surface class I major histocompatibility complex expression by over 90%, whereas RID alpha/beta expression inhibited cytokine-induced Fas upregulation by over 75%. beta TC-tet cells transduced with gp19K and RID alpha/beta lentivectors, but not with a control lentivector, provided prolonged correction of hyperglycemia after transplantation into diabetic BALB/c severe combined immunodeficient mice reconstituted with allogeneic immune effector cells or into diabetic allogeneic BALB/c mice. Thus, genetic engineering of beta-cells using gp19K- and RID alpha/beta-expressing lentiviral vectors may provide an alternative that has the potential to eliminate or reduce treatment with the potent immunosuppressive agents necessary at present for prolonged engraftment with transplanted islets.

  1. Induction of Ski protein expression upon luteinization in rat granulosa cells without a change in its mRNA expression.

    Science.gov (United States)

    Kim, Hyun; Yamanouchi, Keitaro; Matsuwaki, Takashi; Nishihara, Masugi

    2012-01-01

    The Ski protein is implicated in the proliferation/differentiation of a variety of cells. We previously reported that the Ski protein is present in granulosa cells of atretic follicles, but not in preovulatory follicles, suggesting that Ski has a role in apoptosis of granulosa cells. However, granulosa cells cannot only undergo apoptosis but can alternatively differentiate into luteal cells. It is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of the present study was to determine the localization of the Ski protein in the rat ovary during luteinization to examine if Ski might play a role in this process. In order to examine the Ski protein expression during the progression of luteinization, follicular growth was induced in immature female rats by administration of equine chorionic gonadotropin, and luteinization was induced by human chorionic gonadotropin treatment to mimic the luteinizing hormone (LH) surge. While no Ski-positive granulosa cells were present in the preovulatory follicle, Ski protein expression was induced in response to the LH surge and was maintained after formation of the corpus luteum (CL). Although the Ski protein is absent from the granulosa cells of the preovulatory follicle, its mRNA (c-ski) was expressed, and the level of c-ski mRNA was unchanged even after the LH surge. The combined results demonstrated that Ski protein expression is induced in granulosa cells upon luteinization, and suggested that its expression is regulated posttranscriptionally.

  2. Protein kinase CK2: evidence for a protein kinase CK2beta subunit fraction, devoid of the catalytic CK2alpha subunit, in mouse brain and testicles

    DEFF Research Database (Denmark)

    Guerra, B; Siemer, S; Boldyreff, B

    1999-01-01

    signals were observed for lung, liver and testicles. In the case of CK2beta mRNA the highest signals were found for testicles, kidney, brain and liver. The amount of CK2beta mRNA in testicles was estimated to be about 6-fold higher than in brain. The strongest CK2beta signals in the Western blot were...... in brain and testicles. By contrast, Northern blot analyses of the CK2alpha mRNA revealed a somewhat different picture. Here, the strongest signals were obtained for brain, liver, heart and lung. In kidney, spleen and testicles mRNAs were only weakly detectable. For CK2alpha' mRNA distribution strong...

  3. Functional characterization of recombinant rat macrophage inflammatory protein-1 alpha and mRNA expression in pulmonary inflammation.

    Science.gov (United States)

    Shi, M M; Chong, I W; Long, N C; Love, J A; Godleski, J J; Paulauskis, J D

    1998-02-01

    Chemokines are important inflammatory mediators that function by activating and recruiting leukocytes to an inflamed tissue. We have recently cDNA cloned the rat chemokine macrophage inflammatory protein-1 alpha (MIP-1 alpha) (1). In the present study, we characterize the biological function of recombinant MIP-1 alpha protein and describe expression of its mRNA both in vitro and in a rat model of lung inflammation. In vitro rat rMIP-1 alpha protein was chemotactic for both polymorphonuclear leukocytes (PMNs) and macrophages with maximal activity at 50 nM for both cell types. In in vivo studies, we found that intratracheal instillation of 1 and 5 micrograms of rMIP-1 alpha resulted in a significant (P < 0.05) influx of cells, primarily monocytes/macrophages, into the airspace of the lungs after 6 h. Mean numbers of lavagable PMNs were not elevated significantly (P < 0.05) for either dose of MIP-1 alpha. As a model of inflammation, rats were intratracheally instilled with 0.1 mg/kg bacterial lipopolysaccharide (LPS). Bronchoalveolar lavage (BAL) was performed 3 h later. Instillation of LPS resulted in an acute neutrophilia, but no significant change in lavagable macrophages. BAL cells from control animals (saline instilled) displayed no basal mRNA expression of either MIP-1 alpha or MIP-2 (positive control). In contrast, both MIP-1 alpha and MIP-2 mRNA levels increased markedly in BAL cells from rats instilled with LPS. The rat alveolar macrophage cell line (NR8383) also showed increased MIP-1 alpha mRNA levels in response to LPS (10 micrograms/ml) with a maximal increase after 6-8 h. The induction of MIP-1 alpha mRNA expression by LPS in NR8383 cells was attenuated by cotreatment with the antioxidants N-acetylcysteine and dimethylsulfoxide, suggesting that the induction of MIP-1 alpha mRNA by LPS is mediated via the generation of reactive oxygen species. We conclude that MIP-1 alpha is a potent chemoattractant for macrophages in vivo, and its mRNA expression in

  4. The RNA-binding protein HuR regulates DNA methylation through stabilization of DNMT3b mRNA.

    Science.gov (United States)

    López de Silanes, Isabel; Gorospe, Myriam; Taniguchi, Hiroaki; Abdelmohsen, Kotb; Srikantan, Subramanya; Alaminos, Miguel; Berdasco, María; Urdinguio, Rocío G; Fraga, Mario F; Jacinto, Filipe V; Esteller, Manel

    2009-05-01

    The molecular basis underlying the aberrant DNA-methylation patterns in human cancer is largely unknown. Altered DNA methyltransferase (DNMT) activity is believed to contribute, as DNMT expression levels increase during tumorigenesis. Here, we present evidence that the expression of DNMT3b is post-transcriptionally regulated by HuR, an RNA-binding protein that stabilizes and/or modulates the translation of target mRNAs. The presence of a putative HuR-recognition motif in the DNMT3b 3'UTR prompted studies to investigate if this transcript associated with HuR. The interaction between HuR and DNMT3b mRNA was studied by immunoprecipitation of endogenous HuR ribonucleoprotein complexes followed by RT-qPCR detection of DNMT3b mRNA, and by in vitro pulldown of biotinylated DNMT3b RNAs followed by western blotting detection of HuR. These studies revealed that binding of HuR stabilized the DNMT3b mRNA and increased DNMT3b expression. Unexpectedly, cisplatin treatment triggered the dissociation of the [HuR-DNMT3b mRNA] complex, in turn promoting DNMT3b mRNA decay, decreasing DNMT3b abundance, and lowering the methylation of repeated sequences and global DNA methylation. In summary, our data identify DNMT3b mRNA as a novel HuR target, present evidence that HuR affects DNMT3b expression levels post-transcriptionally, and reveal the functional consequences of the HuR-regulated DNMT3b upon DNA methylation patterns.

  5. Pharmacological zinc and phytase supplementation enhance metallothionein mRNA abundance and protein concentration in newly weaned pigs.

    Science.gov (United States)

    Martínez, Michelle M; Hill, Gretchen M; Link, Jane E; Raney, Nancy E; Tempelman, Robert J; Ernst, Catherine W

    2004-03-01

    The swine industry feeds pharmacological zinc (Zn) to newly weaned pigs to improve health. Because most swine diets are plant-based with a high phytic acid content, we hypothesized that adding phytase to diets could reduce the amount of Zn required to obtain beneficial responses. The role of metallothionein (MT) in Zn homeostasis could be important in this positive response. Thus, the goal of this study was to investigate the effect of dietary Zn and phytase on relative MT mRNA abundance and protein concentration in newly weaned pigs. Diets containing adequate (150 mg Zn/kg) or pharmacological concentrations of Zn (1000 or 2000 mg Zn/kg), as zinc oxide, with or without phytase [0, 500 phytase units (FTU)/kg, Natuphos, BASF] were fed in a 3 x 2 factorial design. Plasma and tissue minerals were measured in pigs killed after 14 d of dietary intervention. Hepatic and renal relative MT mRNA abundance and protein were greater (P pigs fed 1000 mg Zn/kg with phytase, or 2000 mg Zn/kg with or without phytase vs. the remaining treatments. Intestinal mucosa MT mRNA abundance and protein were greater (P pigs fed 2000 mg Zn/kg with phytase than in pigs fed 2000 mg Zn/kg alone or 1000 mg Zn/kg with phytase. Pigs fed 1000 mg Zn/kg plus phytase or 2000 mg Zn/kg with or without phytase had higher plasma, hepatic, and renal Zn than those fed the adequate Zn diets or 1000 mg Zn/kg. We conclude that feeding 1000 mg Zn/kg with phytase enhances MT mRNA abundance and protein and Zn absorption to the same degree as 2000 mg Zn/kg with and without phytase.

  6. Analysis of p130 protein and mRNA expression in ten patients with uterine papillary serous carcinoma

    Directory of Open Access Journals (Sweden)

    Shao-ting XU

    2011-11-01

    Full Text Available Objective To examine p130 protein and mRNA expression in uterine papillary serous carcinoma(UPSC and their clinical and pathologic significance.Methods A total of 10 UPSC patients(Stage I were included,with 10 cases of high-level endometrial carcinoma of the same stage taken as the control group and 10 cases of normal proliferative stage endometrium(EM taken as the disease control group.The level of p130 protein expression was determined by hematoxylin and eosin staining,microscopic observation,and immunohistochemistry,whereas the p130 mRNA levels were examined through real-time quantitative reverse transcriptase polymerase chain reaction.The clinicopathologic analysis was carried out in combination with clinical data.Results The p130 protein and p130 mRNA expression levels in the UPSC group(0.46±0.01 and 0.56±0.06,respectively were apparently less than that of the normal proliferative stage endometrium group(0.91±0.04 and 2.81±0.40,respectively;P < 0.01 and also less than those in high-level endometrial carcinoma(P < 0.05.Clinicopathologic analysis shows that all patients are post-menopausal women with symptoms of irregular vaginal bleeding and the average tumor size was 7.5cm(range: 1.2-14.8cm.The pathologic features are same as that of high-level ovarian papillary serous carcinoma.Conclusion Reduced p130 protein and p130 mRNA expression in UPSC might correlate with poor prognosis in UPSC patients.

  7. Position-dependent interactions of Y-box protein 2 (YBX2) with mRNA enable mRNA storage in round spermatids by repressing mRNA translation and blocking translation-dependent mRNA decay.

    Science.gov (United States)

    Kleene, Kenneth C

    2016-03-01

    Many mRNAs encoding proteins needed for the construction of the specialized organelles of spermatozoa are stored as translationally repressed, free messenger ribonucleoproteins in round spermatids, to be actively translated in elongating and elongated spermatids. The factors that repress translation in round spermatids, however, have been elusive. Two lines of evidence implicate the highly abundant and well-known translational repressor, Y-box protein 2 (YBX2), as a critical factor: First, protamine 1 (Prm1) and sperm-mitochondria cysteine-rich protein (Smcp) mRNAs are prematurely recruited onto polysomes in Ybx2-knockout mouse round spermatids. Second, mutations in 3' untranslated region (3'UTR) cis-elements that abrogate YBX2 binding activate translation of Prm1 and Smcp mRNAs in round spermatids of transgenic mice. The abundance of YBX2 and its affinity for variable sequences, however, raise questions of how YBX2 targets specific mRNAs for repression. Mutations to the Prm1 and Smcp mRNAs in transgenic mice reveal that strong repression in round spermatids requires YBX2 binding sites located near the 3' ends of their 3'UTRs as locating the same sites in upstream positions produce negligible repression. This location-dependence implies that the assembly of repressive complexes is nucleated by adjacent cis-elements that enable cooperative interactions of YBX2 with co-factors. The available data suggest that, in vertebrates, YBX2 has the important role of coordinating the storage of translationally repressed mRNAs in round spermatids by inhibiting translational activity and the degradation of transcripts via translation-dependent deadenylation. These insights should facilitiate future experiments designed to unravel how YBX2 targets mRNAs for repression in round spermatids and how mutations in the YBX2 gene cause infertility in humans. Mol. Reprod. Dev. 83: 190-207, 2016. © 2016 Wiley Periodicals, Inc.

  8. AU-RICH ELEMENTS IN THE 3′-UTR REGULATE THE STABILITY OF THE 141 AMINO ACID ISOFORM OF PARATHYROID HORMONE-RELATED PROTEIN mRNA

    Science.gov (United States)

    Luchin, Alexander I.; Nadella, Murali V.P.; Thudi, Nanda K.; Dirksen, Wessel P.; Gulati, Parul; Fernandez, Soledad A.; Rosol, Thomas J.

    2012-01-01

    We demonstrated previously that parathyroid hormone-related protein (PTHrP) 1-141 mRNA is the least stable of three isoforms and is the only isoform that is stabilized by TGF-β. In order to understand how PTHrP mRNA is stabilized by TGF-β, we first sought to elucidate the mechanism(s) that are responsible for the instability of PTHrP isoform 1-141 mRNA. The 3′-UTR of isoform 1-141 contains four AU-rich elements (AREs), which are known to mediate mRNA degradation. We utilized a luciferase reporter system to test whether these four AREs are responsible for the short half-life of PTHrP 1-141 mRNA. Our results demonstrated that ARE elements in the 3′-UTR of PTHrP 1-141 mRNA play a significant role in regulation of the stability of the mRNA. It is known that AREs mediate their effects on mRNA stability through a number of ARE-binding proteins that recruit the exosome, a complex of exonucleases that degrades the mRNA. We identified tristetraproline (TTP) as an RNA-binding protein that may be involved in ARE-mediated degradation of PTHrP 1-141 mRNA. PMID:22960231

  9. AU-rich elements in the 3'-UTR regulate the stability of the 141 amino acid isoform of parathyroid hormone-related protein mRNA.

    Science.gov (United States)

    Luchin, Alexander I; Nadella, Murali V P; Thudi, Nanda K; Dirksen, Wessel P; Gulati, Parul; Fernandez, Soledad A; Rosol, Thomas J

    2012-11-25

    We demonstrated previously that parathyroid hormone-related protein (PTHrP) 1-141 mRNA is the least stable of three isoforms and is the only isoform that is stabilized by TGF-β. In order to understand how PTHrP mRNA is stabilized by TGF-β, we first sought to elucidate the mechanism(s) that are responsible for the instability of PTHrP isoform 1-141 mRNA. The 3'-UTR of isoform 1-141 contains four AU-rich elements (AREs), which are known to mediate mRNA degradation. We utilized a luciferase reporter system to test whether these four AREs are responsible for the short half-life of PTHrP 1-141 mRNA. Our results demonstrated that ARE elements in the 3'-UTR of PTHrP 1-141 mRNA play a significant role in regulation of the stability of the mRNA. It is known that AREs mediate their effects on mRNA stability through a number of ARE-binding proteins that recruit the exosome, a complex of exonucleases that degrades the mRNA. We identified tristetraproline (TTP) as an RNA-binding protein that may be involved in ARE-mediated degradation of PTHrP 1-141 mRNA. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  10. Transforming growth factor-beta 1 specifically induce proteins involved in the myofibroblast contractile apparatus

    DEFF Research Database (Denmark)

    Malmström, Johan; Lindberg, Henrik Have; Lindberg, Claes

    2004-01-01

    ) alters protein expression of cytoskeletal-associated proteins. Metabolic labeling of cell cultures by [(35)S]methionine, followed by protein separation on two-dimensional gel electrophoresis, displayed approximately 2500 proteins in the pI interval of 3-10. Treatment of TGF-beta(1) led to specific spot...... expression of alpha-SMA and may participate in the formation of stress fibers, cell contractility, and cell spreading characterizing the myofibroblasts phenotype....

  11. Expression of fragile X mental retardation protein and Fmr1 mRNA during folliculogenesis in the rat.

    Science.gov (United States)

    Ferder, Ianina; Parborell, Fernanda; Sundblad, Victoria; Chiauzzi, Violeta; Gómez, Karina; Charreau, Eduardo H; Tesone, Marta; Dain, Liliana

    2013-04-01

    Fragile X mental retardation protein (FMRP) belongs to a small family of RNA-binding proteins. Its absence or inactivity is responsible for fragile X syndrome, the most common cause of inherited mental retardation. Despite its ubiquitous expression, FMRP function and expression remain almost understudied in non-neuronal tissues, though previous studies on germline development during oogenesis may suggest a special function of this protein also in ovarian tissue. In addition, the well-documented association of FMR1 premutation state with fragile X-related premature ovarian insufficiency adds interest to the role of FMRP in ovarian physiology. The aim of the present work was to investigate the expression of Fmr1 mRNA and its protein, FMRP, at different stages of rat follicular development. By immunohistochemical studies we demonstrated FMRP expression in granulosa, theca and germ cells in all stages of follicular development. In addition, changes in Fmr1 expression, both at the protein and mRNA levels, were observed. FMRP levels increased upon follicular development while preantral and early antral follicles presented similar levels of Fmr1 transcripts with decreased expression in preovulatory follicles. These observations suggest that Fmr1 expression in the ovary is regulated at different and perhaps independent levels. In addition, our results show expression of at least four different isoforms of FMRP during all stages of follicular growth with expression patterns that differ from those observed in brain and testis. Our study shows a regulated expression of Fmr1, both at mRNA and protein levels, during rat follicular development.

  12. Combinatorial Control of mRNA Fates by RNA-Binding Proteins and Non-Coding RNAs

    Directory of Open Access Journals (Sweden)

    Valentina Iadevaia

    2015-09-01

    Full Text Available Post-transcriptional control of gene expression is mediated by RNA-binding proteins (RBPs and small non-coding RNAs (e.g., microRNAs that bind to distinct elements in their mRNA targets. Here, we review recent examples describing the synergistic and/or antagonistic effects mediated by RBPs and miRNAs to determine the localisation, stability and translation of mRNAs in mammalian cells. From these studies, it is becoming increasingly apparent that dynamic rearrangements of RNA-protein complexes could have profound implications in human cancer, in synaptic plasticity, and in cellular differentiation.

  13. Stabilization of TRAIL, an all-{beta}-sheet multimeric protein, using computational redesign

    NARCIS (Netherlands)

    van der Sloot, Almer Martinus; Mullally, Margaret; Fernandez-Ballester, G.; Serrano, L.; Quax, Wim

    2004-01-01

    Protein thermal stability is important for therapeutic proteins, both influencing the pharmacokinetic and pharmacodynamic properties and for stability during production and shelf-life of the final product. In this paper we show the redesign of a therapeutically interesting trimeric all-beta-sheet pr

  14. Stabilization of TRAIL, an all-beta-sheet multimeric protein, using computational redesign

    NARCIS (Netherlands)

    van der Sloot, AM; Mullally, MM; Fernandez-Ballester, G; Serrano, L; Quax, WJ

    2004-01-01

    Protein thermal stability is important for therapeutic proteins, both influencing the pharmacokinetic and pharmacodynamic properties and for stability during production and shelf-life of the final product. In this paper we show the redesign of a therapeutically interesting trimeric all-beta-sheet pr

  15. The beta-barrel outer membrane protein assembly complex of Neisseria meningitidis.

    NARCIS (Netherlands)

    Volokhina, E.B.; Beckers, F.; Tommassen, J.; Bos, M.P.

    2009-01-01

    The evolutionarily conserved protein Omp85 is required for outer membrane protein (OMP) assembly in gram-negative bacteria and in mitochondria. Its Escherichia coli homolog, designated BamA, functions with four accessory lipoproteins, BamB, BamC, BamD, and BamE, together forming the beta-barrel asse

  16. The Disulfide Bond Pattern of Transforming Growth Factor Beta-Induced protein

    DEFF Research Database (Denmark)

    Lukassen, Marie V; Scavenius, Carsten; Thøgersen, Ida B;

    2016-01-01

    Transforming growth factor beta-induced protein (TGFBIp) is an extracellular matrix protein composed of an NH2-terminal cysteine-rich domain (CRD) annotated as an emilin (EMI) domain, and four fasciclin-1 (FAS1-1 to FAS1-4) domains. Mutations in the gene cause corneal dystrophies, a group...

  17. Detection of tumor cell-specific mRNA and protein in exosome-like microvesicles from blood and saliva.

    Science.gov (United States)

    Yang, Jieping; Wei, Fang; Schafer, Christopher; Wong, David T W

    2014-01-01

    The discovery of disease-specific biomarkers in oral fluids has revealed a new dimension in molecular diagnostics. Recent studies have reported the mechanistic involvement of tumor cells derived mediators, such as exosomes, in the development of saliva-based mRNA biomarkers. To further our understanding of the origins of disease-induced salivary biomarkers, we here evaluated the hypothesis that tumor-shed secretory lipidic vesicles called exosome-like microvesicles (ELMs) that serve as protective carriers of tissue-specific information, mRNAs, and proteins, throughout the vasculature and bodily fluids. RNA content was analyzed in cell free-saliva and ELM-enriched fractions of saliva. Our data confirmed that the majority of extracellular RNAs (exRNAs) in saliva were encapsulated within ELMs. Nude mice implanted with human lung cancer H460 cells expressing hCD63-GFP were used to follow the circulation of tumor cell specific protein and mRNA in the form of ELMs in vivo. We were able to identify human GAPDH mRNA in ELMs of blood and saliva of tumor bearing mice using nested RT-qPCR. ELMs positive for hCD63-GFP were detected in the saliva and blood of tumor bearing mice as well as using electric field-induced release and measurement (EFIRM). Altogether, our results demonstrate that ELMs carry tumor cell-specific mRNA and protein from blood to saliva in a xenografted mouse model of human lung cancer. These results therefore strengthen the link between distal tumor progression and the biomarker discovery of saliva through the ELMs.

  18. Posttranscriptional regulation of GAP-43 gene expression in PC12 cells through protein kinase C-dependent stabilization of the mRNA.

    Science.gov (United States)

    Perrone-Bizzozero, N I; Cansino, V V; Kohn, D T

    1993-03-01

    We have previously shown that nerve growth factor (NGF) selectively stabilizes the GAP-43 mRNA in PC12 cells. To study the cellular mechanisms for this post-transcriptional control and to determine the contribution of mRNA stability to GAP-43 gene expression, we examined the effects of several agents that affect PC12 cell differentiation on the level of induction and rate of degradation of the GAP-43 mRNA. The NGF-mediated increase in GAP-43 mRNA levels and neurite outgrowth was mimicked by the phorbol ester TPA, but not by dibutyryl cAMP or the calcium ionophore A12783. Downregulation of protein kinase C (PKC) by high doses of phorbol esters or selective PKC inhibitors prevented the induction of this mRNA by NGF, suggesting that NGF and TPA act through a common PKC-dependent pathway. In mRNA decay studies, phorbol esters caused a selective 6-fold increase in the half-life of the GAP-43 mRNA, which accounts for most of the induction of this mRNA by TPA. The phorbol ester-induced stabilization of GAP-43 mRNA was blocked by the protein kinase inhibitor polymyxin B and was partially inhibited by dexamethasone, an agent that blocks GAP-43 expression and neuronal differentiation in PC12 cells. In contrast, the rates of degradation and the levels of the GAP-43 mRNA in control and TPA-treated cells were not affected by cycloheximide treatment. Thus, changes in GAP-43 mRNA turnover do not appear to require continuous protein synthesis. In conclusion, these data suggest that PKC activity regulates the levels of the GAP-43 mRNA in PC12 cells through a novel, translation-independent mRNA stabilization mechanism.

  19. Analysis of MDM2 and MDM4 single nucleotide polymorphisms, mRNA splicing and protein expression in retinoblastoma.

    Directory of Open Access Journals (Sweden)

    Justina McEvoy

    Full Text Available Retinoblastoma is a childhood cancer of the developing retina that begins in utero and is diagnosed in the first years of life. Biallelic RB1 gene inactivation is the initiating genetic lesion in retinoblastoma. The p53 gene is intact in human retinoblastoma but the pathway is believed to be suppressed by increased expression of MDM4 (MDMX and MDM2. Here we quantify the expression of MDM4 and MDM2 mRNA and protein in human fetal retinae, primary retinoblastomas, retinoblastoma cell lines and several independent orthotopic retinoblastoma xenografts. We found that MDM4 is the major p53 antagonist expressed in retinoblastoma and in the developing human retina. We also discovered that MDM4 protein steady state levels are much higher in retinoblastoma than in human fetal retinae. This increase would not have been predicted based on the mRNA levels. We explored several possible post-transcriptional mechanisms that may contribute to the elevated levels of MDM4 protein. A proportion of MDM4 transcripts are alternatively spliced to produce protein products that are reported to be more stable and oncogenic. We also discovered that a microRNA predicted to target MDM4 (miR191 was downregulated in retinoblastoma relative to human fetal retinae and a subset of samples had somatic mutations that eliminated the miR-191 binding site in the MDM4 mRNA. Taken together, these data suggest that post-transcriptional mechanisms may contribute to stabilization of the MDM4 protein in retinoblastoma.

  20. Monitoring mRNA and protein levels in bulk and in model vesicle-based artificial cells.

    Science.gov (United States)

    van Nies, Pauline; Canton, Alicia Soler; Nourian, Zohreh; Danelon, Christophe

    2015-01-01

    With rising interest in utilizing cell-free gene expression systems in bottom-up synthetic biology projects, novel labeling tools need to be developed to accurately report the dynamics and performance of the biosynthesis machinery operating in various reaction conditions. Monitoring the transcription activity has been simplified by the Spinach technology, an RNA aptamer that emits fluorescence upon binding to a small organic dye. Recently, we tracked the fluorescence of Spinach-tagged messenger RNA (mRNA) and its translation product the yellow fluorescent protein (YFP), both synthesized in the protein synthesis using recombinant elements system from a DNA template. Building on our previous study, we describe here an improved Spinach reporter with modified flanking sequences that confer higher propensity for aptamer folding and, thus, enhanced fluorescence brightness. Hence, the kinetics of mRNA and YFP production could be simultaneously monitored with unprecedented sensitivity. A combination of methodologies, comprising RNA gel analysis, real-time quantitative polymerase chain reaction, absorbance measurements, and fluorescence correlation spectroscopy, was used to convert fluorescence intensity units into absolute concentrations of transcript and YFP translational product. Furthermore, we demonstrated that the new Spinach construct greatly enhanced mRNA detection when gene expression was confined inside self-assembled lipid vesicles. Therefore, we argue that this assay could be used to evaluate systematically the performance of transcription and translation in model vesicle-based artificial cells.

  1. Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

    DEFF Research Database (Denmark)

    Beuchert Kallehauge, Thomas; Li, Shangzhong; Pedersen, Lasse Ebdrup

    2017-01-01

    as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we......Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated...... as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated...

  2. Traffic of interacting ribosomes on mRNA during protein synthesis: effects of chemo-mechanics of individual ribosomes

    CERN Document Server

    Basu, A; Basu, Aakash; Chowdhury, Debashish

    2006-01-01

    Many {\\it ribosomes} simultaneously move on the same messenger RNA (mRNA), each synthesizing a protein. In contrast to the earlier models, here {\\it we develope a ``unified'' theoretical model} that not only incorporates the {\\it mutual exclusions} of the interacting ribosomes, but also describes explicitly the mechano-chemistry of each of these individual cyclic machines during protein synthesis. Using a combination of analytical and numerical techniques of non-equilibrium statistical mechanics, we analyze the rates of protein synthesis and the spatio-temporal oraganization of the ribosomes in this model. We also predict how these properties would change with the changes in the rates of the various chemo-mechanical processes in each ribosome. Finally, we illustrate the power of this model by making experimentally testable predictions on the rates of protein synthesis and the density profiles of the ribosomes on some mRNAs in {\\it E-coli}.

  3. mRNA and protein dataset of autophagy markers (LC3 and p62 in several cell lines

    Directory of Open Access Journals (Sweden)

    Rubén Gómez-Sánchez

    2016-06-01

    Full Text Available We characterized the dynamics of autophagy in vitro using four different cell systems and analyzing markers widely used in this field, i.e. LC3 (microtubule-associated protein 1 light chain 3; protein recruited from the cytosol (LC3-I to the autophagosomal membrane where it is lipidated (LC3-II and p62/SQSTM1 (adaptor protein that serves as a link between LC3 and ubiquitinated substrates, (Klionsky et al., 2016 [1]. Data provided include analyses of protein levels of LC3 and p62 by Western-blotting and endogenous immunofluorescence experiments, but also p62 mRNA levels obtained by quantitative PCR (qPCR. To monitor the turnover of these autophagy markers and, thus, measure the flux of this pathway, cells were under starvation conditions and/or treated with bafilomycin A1 (Baf. A1 to block fusion of autophagosomes with lysosomes.

  4. mRNA and protein dataset of autophagy markers (LC3 and p62) in several cell lines

    Science.gov (United States)

    Gómez-Sánchez, Rubén; Yakhine-Diop, Sokhna M.S.; Rodríguez-Arribas, Mario; Bravo-San Pedro, José M.; Martínez-Chacón, Guadalupe; Uribe-Carretero, Elisabet; Pinheiro de Castro, Diana C.J.; Pizarro-Estrella, Elisa; Fuentes, José M.; González-Polo, Rosa A.

    2016-01-01

    We characterized the dynamics of autophagy in vitro using four different cell systems and analyzing markers widely used in this field, i.e. LC3 (microtubule-associated protein 1 light chain 3; protein recruited from the cytosol (LC3-I) to the autophagosomal membrane where it is lipidated (LC3-II)) and p62/SQSTM1 (adaptor protein that serves as a link between LC3 and ubiquitinated substrates), (Klionsky et al., 2016) [1]. Data provided include analyses of protein levels of LC3 and p62 by Western-blotting and endogenous immunofluorescence experiments, but also p62 mRNA levels obtained by quantitative PCR (qPCR). To monitor the turnover of these autophagy markers and, thus, measure the flux of this pathway, cells were under starvation conditions and/or treated with bafilomycin A1 (Baf. A1) to block fusion of autophagosomes with lysosomes. PMID:27054171

  5. Luteotropic and luteolytic factors regulate mRNA and protein expression of progesterone receptor isoforms A and B in the bovine endometrium.

    Science.gov (United States)

    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.

  6. Steady-state concentrations of mRNA encoding two inhibitors of protein kinase C in ovine luteal tissue.

    Science.gov (United States)

    Juengel, J L; Melner, M H; Clapper, J A; Turzillo, A M; Moss, G E; Nett, T M; Niswender, G D

    1998-07-01

    Prostaglandin F2 alpha (PGF2 alpha) decreases secretion of progesterone from the corpus luteum in domestic ruminants. However, it is less effective during the early part of the oestrous cycle (Louis et al., 1973) and at the time of maternal recognition of pregnancy (Silvia and Niswender, 1984; Lacroix and Kann, 1986). Decreased luteal responsiveness may be due to failure of PGF2 alpha to activate fully its normal second messenger system, protein kinase C (PKC). Alternatively, increased resistance of the corpus luteum to PGF2 alpha might be attributable to greater concentrations of recently identified biological inhibitors of PKC. These possibilities were addressed by measuring steady-state concentrations of mRNA encoding PGF2 alpha receptor and two inhibitors of PKC, protein kinase C inhibitor-1 (PKCI-1) and kinase C inhibitor protein-1 (KCIP-1, brain 14-3-3 protein), in corpora lutea collected from ewes on days 4, 10 and 15 of the oestrous cycle (n = 5 per day) and day 15 of pregnancy (n = 7). There were no differences in mean concentrations of mRNA encoding PGF2 alpha receptor among the groups. However, concentrations of mRNA encoding both inhibitors of PKC were higher (P day 4 of the oestrous cycle compared with the other groups. Treatment of ewes with a luteolytic dose of PGF2 alpha, which activates PKC, did not change concentrations of mRNA encoding either PKCI-1 or KCIP-I up to 24 h later. Luteal expression of mRNA encoding the PKC inhibitors and PGF2 alpha receptor was also examined in ewes treated with oestradiol in vivo for 16 h in the midluteal phase. High concentrations of oestradiol in serum (20 and 70 pg ml-1) did not influence quantities of any of the mRNAs examined. Therefore, an increase in PKC inhibitors may be involved in resistance of the corpus luteum to PGF2 alpha during the early part of the oestrous cycle but does not appear to mediate the increased resistance of the corpus luteum to PGF2 alpha during maternal recognition of pregnancy

  7. Osteocalcin and matrix GLA protein in developing teleost teeth: identification of sites of mRNA and protein accumulation at single cell resolution.

    Science.gov (United States)

    Ortiz-Delgado, J B; Simes, D C; Gavaia, P; Sarasquete, C; Cancela, M L

    2005-08-01

    In this study, the tissue distribution and accumulation of osteocalcin or bone Gla protein (BGP) and matrix Gla protein (MGP) were determined during tooth development in a teleost fish, Argyrosomus regius. In this species, the presence of BGP and MGP mRNA in teeth was revealed by in situ hybridization. mRNA for BGP was detected in the odontoblasts as well as in its cytoplasmic processes emerging through dentinal tubules, while mRNA for MGP was expressed in the enamel portion within the apical portion of the elongated cell bodies of enameloblasts, adjacent to the root of the teeth as well as in cells within the pulpal space. Immunolocalization of BGP and MGP demonstrated that these proteins accumulate mainly in the mineralized dentin or in enameloblastic processes, confirming in situ hybridization results. In this study, we examined for the first time the localization of both BGP and MGP gene expression and protein accumulation within the different regions of the vertebrate tooth. We clearly demonstrated that although the overall pattern of BGP and MGP gene expression and protein accumulation in A. regius teeth was in general agreement to what is known for other vertebrates such as rats or rodents, our study provided novel information and highlighted some species-differences between fish and higher vertebrates.

  8. Cytokine production in the central nervous system of Lewis rats with experimental autoimmune encephalomyelitis: dynamics of mRNA expression for interleukin-10, interleukin-12, cytolysin, tumor necrosis factor alpha and tumor necrosis factor beta

    DEFF Research Database (Denmark)

    Issazadeh-Navikas, Shohreh; Ljungdahl, A; Höjeberg, B

    1995-01-01

    in cryosections of spinal cords using in situ hybridization technique with synthetic oligonucleotide probes. Three stages of cytokine mRNA expression could be distinguished: (i) interleukin (IL)-12, tumor necrosis factor (TNF)-beta (= lymphotoxin-alpha) and cytolysin appeared early and before onset of clinical...... signs of EAE; (ii) TNF-alpha peaked at height of clinical signs of EAE; (iii) IL-10 appeared increasingly at and after clinical recovery. The early expression of IL-12 prior to the expression of interferon-gamma (IFN-gamma) mRNA shown previously is consistent with a role of IL-12 in promoting...

  9. Characterization of the beta amyloid precursor protein-like gene in the central nervous system of the crab Chasmagnathus. Expression during memory consolidation

    Directory of Open Access Journals (Sweden)

    Fustiñana Maria

    2010-09-01

    Full Text Available Abstract Background Human β-amyloid, the main component in the neuritic plaques found in patients with Alzheimer's disease, is generated by cleavage of the β-amyloid precursor protein. Beyond the role in pathology, members of this protein family are synaptic proteins and have been associated with synaptogenesis, neuronal plasticity and memory, both in vertebrates and in invertebrates. Consolidation is necessary to convert a short-term labile memory to a long-term and stable form. During consolidation, gene expression and de novo protein synthesis are regulated in order to produce key proteins for the maintenance of plastic changes produced during the acquisition of new information. Results Here we partially cloned and sequenced the beta-amyloid precursor protein like gene homologue in the crab Chasmagnathus (cappl, showing a 37% of identity with the fruit fly Drosophila melanogaster homologue and 23% with Homo sapiens but with much higher degree of sequence similarity in certain regions. We observed a wide distribution of cappl mRNA in the nervous system as well as in muscle and gills. The protein localized in all tissues analyzed with the exception of muscle. Immunofluorescence revealed localization of cAPPL in associative and sensory brain areas. We studied gene and protein expression during long-term memory consolidation using a well characterized memory model: the context-signal associative memory in this crab species. mRNA levels varied at different time points during long-term memory consolidation and correlated with cAPPL protein levels Conclusions cAPPL mRNA and protein is widely distributed in the central nervous system of the crab and the time course of expression suggests a role of cAPPL during long-term memory formation.

  10. Atualizações sobre beta-hidroxi-beta-metilbutirato: suplementação e efeitos sobre o catabolismo de proteínas New findings on beta-hydroxy-beta-methylbutyirate: supplementation and effects on the protein catabolism

    Directory of Open Access Journals (Sweden)

    Everson Araújo Nunes

    2008-04-01

    Full Text Available O beta-hidroxi-beta-metilbutirato, metabólito do aminoácido leucina, vem sendo utilizado como suplemento alimentar, em situações específicas, com o intuito de aumentar ou manter a massa isenta de gordura. Os relatos dos efeitos do beta-hidroxi-beta-metilbutirato em estudos recentes fizeram crescer as expectativas sobre sua utilização em casos patológicos. Também foram demonstrados melhores resultados, quando da sua ingestão, no treinamento de força em indivíduos iniciantes e em idosos. Em humanos o beta-hidroxi-beta-metilbutirato tem sido usado como agente anti-catabólico, e em modelos animais foi demonstrado ser eficaz em inibir a atividade de vias proteolíticas em células musculares de indivíduos caquéticos in vitro e in vivo. Os mecanismos participantes desses processos envolvem: a inibição da atividade do sistema ubiquitina proteossoma ATP-dependente, a inibição de vias de sinalização com participação da proteína quinase C-alfa e a diminuição da concentração citoplasmática do fator nuclear - kappa B livre, eventos relacionados ao decréscimo da proteólise em células musculares.The leucine metabolite beta-hydroxy-beta-methylbutyrate has been used as a nutritional supplement in specific situations to prevent losing or to increase lean mass. Recent studies showed interesting results of beta-hydroxy-beta-methylbutyrate supplementation in certain disease states. Better results have also been demonstrated when it is taken by starters or old individuals doing strength training. In humans, beta-hydroxy-beta-methylbutyrate has been used as an anticatabolic agent and in animal models it has been demonstrated to be effective in inhibiting the activity of the proteolytic pathways in muscle cells of extremely weak individuals in vivo and in vitro. The mechanisms that participate in this process involve: inhibition of the ATP-ubiquitin-proteasome pathway, inhibition of the signalization pathways involving protein kinase C

  11. Quantitative protein and fat metabolism in bull calves treated with beta-adrenergic agonist

    DEFF Research Database (Denmark)

    Chwalibog, André; Jensen, K; Thorbek, G

    1996-01-01

    Protein and energy utilization and quantitative retention of protein, fat and energy was investigated with 12 Red Danish bulls during two subsequent 6 weeks trials (Sections A and B) at a mean live weight of 195 and 335 kg respectively. Treatments were control (Group 1) and beta-agonist (L-644...... matter, metabolizable energy and digestible protein was of the same magnitude for all groups. The beta-agonist had no significant effect on protein digestibility and metabolizability of energy, but daily live weight gain was significantly higher in the treated bulls. The utilization of digested protein...... was strongly influenced by treatment, with the highest values for Group 2 in both sections. The protein retention increased with 25% in Group 2, with the highest increment of 113 g/d in Section B. The fat retention decreased in treated animals, most pronounced in Group 3, where the reduction was about 50...

  12. La-related protein 1 (LARP1) represses terminal oligopyrimidine (TOP) mRNA translation downstream of mTOR complex 1 (mTORC1)

    DEFF Research Database (Denmark)

    Fonseca, Bruno; Zakaria, Chadi; Jia, J J

    2015-01-01

    to repress TOP mRNA translation; and (iv) LARP1 competes with the eukaryotic initiation factor (eIF) 4G for TOP mRNA binding. Importantly, from a drug resistance standpoint, our data also show that reducing LARP1 protein levels by RNA interference attenuates the inhibitory effect of rapamycin, Torin1...

  13. Induction of anti-beta(2)-glycoprotein I autoantibodies in mice by protein H of Streptococcus pyogenes

    NARCIS (Netherlands)

    Van Os, G. M. A.; Meijers, J. C. M.; Agar, C.; Seron, M. V.; Marquart, J. A.; Akesson, P.; Urbanus, R. T.; Derksen, R. H. W. M.; Herwald, H.; Morgelin, M.; De Groot, P. G.

    2011-01-01

    Background: The antiphospholipid syndrome (APS) is characterized by the persistent presence of anti-beta 2-glycoprotein I (beta 2-GPI) autoantibodies. beta 2-GPI can exist in two conformations. In plasma it is a circular protein, whereas it adopts a fish-hook conformation after binding to phospholip

  14. Structural and biological mimicry of protein surface recognition by [alpha/beta]-peptide foldamers

    Energy Technology Data Exchange (ETDEWEB)

    Horne, W. Seth; Johnson, Lisa M.; Ketas, Thomas J.; Klasse, Per Johan; Lu, Min; Moore, John P.; Gellman, Samuel H.; (Cornell); (UW)

    2009-10-05

    Unnatural oligomers that can mimic protein surfaces offer a potentially useful strategy for blocking biomedically important protein-protein interactions. Here we evaluate an approach based on combining {alpha}- and {beta}-amino acid residues in the context of a polypeptide sequence from the HIV protein gp41, which represents an excellent testbed because of the wealth of available structural and biological information. We show that {alpha}/{beta}-peptides can mimic structural and functional properties of a critical gp41 subunit. Physical studies in solution, crystallographic data, and results from cell-fusion and virus-infectivity assays collectively indicate that the gp41-mimetic {alpha}/{beta}-peptides effectively block HIV-cell fusion via a mechanism comparable to that of gp41-derived {alpha}-peptides. An optimized {alpha}/{beta}-peptide is far less susceptible to proteolytic degradation than is an analogous {alpha}-peptide. Our findings show how a two-stage design approach, in which sequence-based {alpha} {yields} {beta} replacements are followed by site-specific backbone rigidification, can lead to physical and biological mimicry of a natural biorecognition process.

  15. Self-assembly of the beta2-microglobulin NHVTLSQ peptide using a coarse-grained protein model reveals a beta-barrel species.

    Science.gov (United States)

    Song, Wei; Wei, Guanghong; Mousseau, Normand; Derreumaux, Philippe

    2008-04-10

    Although a wide variety of proteins can assemble into amyloid fibrils, the structure of the early oligomeric species on the aggregation pathways remains unknown at an atomic level of detail. In this paper we report, using molecular dynamics simulations with the OPEP coarse-grained force field, the free energy landscape of a tetramer and a heptamer of the beta2-microglobulin NHVTLSQ peptide. On the basis of a total of more than 17 ns trajectories started from various states, we find that both species are in equilibrium between amorphous and well-ordered aggregates with cross-beta-structure, a perpendicular bilayer beta-sheet, and, for the heptamer, six- or seven-stranded closed and open beta-barrels. Moreover, analysis of the heptamer trajectories shows that the perpendicular bilayer beta-sheet is one possible precursor of the beta-barrel, but that this barrel can also be formed from a twisted monolayer beta-sheet with successive addition of chains. Comparison with previous aggregation simulations and the fact that nature constructs transmembrane beta-sheet proteins with pores open the possibility that beta-barrels with small inner diameters may represent a common intermediate during the early steps of aggregation.

  16. Basal and inducible CYP1 mRNA quantitation and protein localization throughout the mouse gastrointestinal tract.

    Science.gov (United States)

    Uno, Shigeyuki; Dragin, Nadine; Miller, Marian L; Dalton, Timothy P; Gonzalez, Frank J; Nebert, Daniel W

    2008-02-15

    The CYP1A1, CYP1A2, and CYP1B1 enzymes are inducible by benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); metabolism of BaP by these enzymes leads to electrophilic intermediates and genotoxicity. Throughout the gastrointestinal (GI) tract, we systematically compared basal and inducible levels of the CYP1 mRNAs by Q-PCR, and localized the CYP1 proteins by immunohistochemistry. Cyp1(+/+) wild-type were compared with the Cyp1a1(-/-), Cyp1a2(-/-), and Cyp1b1(-/-) single-knockout and Cyp1a1/1b1(-/-) and Cyp1a2/1b1(-/-) double-knockout mice. Oral BaP was compared with intraperitoneal TCDD. In general, maximal CYP1A1 mRNA levels were 3-10 times greater than CYP1B1, which were 3-10 times greater than CYP1A2 mRNA levels. Highest inducible concentrations of CYP1A1 and CYP1A2 occurred in proximal small intestine, whereas the highest basal and inducible levels of CYP1B1 mRNA occurred in esophagus, forestomach, and glandular stomach. Ablation of either Cyp1a2 or Cyp1b1 gene resulted in a compensatory increase in CYP1A1 mRNA - but only in small intestine. Also in small intestine, although BaP- and TCDD-mediated CYP1A1 inductions were roughly equivalent, oral BaP-mediated CYP1A2 mRNA induction was approximately 40-fold greater than TCDD-mediated CYP1A2 induction. CYP1B1 induction by TCDD in Cyp1(+/+) and Cyp1a2(-/-) mice was 4-5 times higher than that by BaP; however, in Cyp1a1(-/-) animals CYP1B1 induction by TCDD or BaP was approximately equivalent. CYP1A1 and CYP1A2 proteins were generally localized nearer to the lumen than CYP1B1 proteins, in both squamous and glandular epithelial cells. These GI tract data suggest that the inducible CYP1A1 enzyme, both in concentration and in location, might act as a "shield" in detoxifying oral BaP and, hence, protecting the animal.

  17. Phosphorylation of the regulatory beta-subunit of protein kinase CK2 by checkpoint kinase Chk1: identification of the in vitro CK2beta phosphorylation site

    DEFF Research Database (Denmark)

    Kristensen, Lars P; Larsen, Martin Røssel; Højrup, Peter;

    2004-01-01

    The regulatory beta-subunit of protein kinase CK2 mediates the formation of the CK2 tetrameric form and it has functions independent of CK2 catalytic subunit through interaction with several intracellular proteins. Recently, we have shown that CK2beta associates with the human checkpoint kinase Chk...... by the modification of Thr213 but it does require the presence of an active Chk1 kinase....

  18. Beta-lactam resistance response triggered by inactivation of a nonessential penicillin-binding protein.

    Directory of Open Access Journals (Sweden)

    Bartolomé Moya

    2009-03-01

    Full Text Available It has long been recognized that the modification of penicillin-binding proteins (PBPs to reduce their affinity for beta-lactams is an important mechanism (target modification by which Gram-positive cocci acquire antibiotic resistance. Among Gram-negative rods (GNR, however, this mechanism has been considered unusual, and restricted to clinically irrelevant laboratory mutants for most species. Using as a model Pseudomonas aeruginosa, high up on the list of pathogens causing life-threatening infections in hospitalized patients worldwide, we show that PBPs may also play a major role in beta-lactam resistance in GNR, but through a totally distinct mechanism. Through a detailed genetic investigation, including whole-genome analysis approaches, we demonstrate that high-level (clinical beta-lactam resistance in vitro, in vivo, and in the clinical setting is driven by the inactivation of the dacB-encoded nonessential PBP4, which behaves as a trap target for beta-lactams. The inactivation of this PBP is shown to determine a highly efficient and complex beta-lactam resistance response, triggering overproduction of the chromosomal beta-lactamase AmpC and the specific activation of the CreBC (BlrAB two-component regulator, which in turn plays a major role in resistance. These findings are a major step forward in our understanding of beta-lactam resistance biology, and, more importantly, they open up new perspectives on potential antibiotic targets for the treatment of infectious diseases.

  19. Cytoplasmic polyadenylation-element-binding protein (CPEB)1 and 2 bind to the HIF-1alpha mRNA 3'-UTR and modulate HIF-1alpha protein expression.

    Science.gov (United States)

    Hägele, Sonja; Kühn, Uwe; Böning, Melanie; Katschinski, Dörthe M

    2009-01-01

    The heterodimeric HIF (hypoxia-inducible factor)-1 is a transcriptional master regulator of several genes involved in mammalian oxygen homoeostasis. Besides the well described regulation of the HIF-1alpha subunit via hydroxylation-mediated protein stability in hypoxia, there are several indications of an additional translational control of the HIF-1alpha mRNA, especially after growth factor stimulation. We identified an interaction of CPEB (cytoplasmic polyadenylation-element-binding protein) 1 and CPEB2 with the 3'-UTR (untranslated region) of HIF-1alpha mRNA. Overexpression of CPEB1 and CPEB2 affected HIF-1alpha protein levels mediated by the 3'-UTR of HIF-1alpha mRNA. Stimulation of neuroblastoma SK-N-MC cells with insulin and thus activation of endogenous CPEBs increased the expression of a luciferase reporter gene fused to the 3'-UTR of HIF-1alpha as well as endogenous HIF-1alpha protein levels. This could be abrogated by treating the cells with CPEB1 or CPEB2 siRNAs (short interfering RNAs). Injection of HIF-1alpha cRNA into Xenopus oocytes verified the elongation of the poly(A)+ (polyadenylated) tail by cytoplasmic polyadenylation. Thus CPEB1 and CPEB2 are involved in the regulation of HIF-1alpha following insulin stimulation.

  20. Nerve growth factor treatment of sensory neuron primary cultures causes elevated levels of the mRNA encoding the ATP synthase beta-subunit as detected by a novel PCR-based differential cloning method.

    Science.gov (United States)

    Kendall, G; Ensor, E; Crankson, H D; Latchman, D S

    1996-03-01

    The mRNA encoding the rat ATP synthase beta-subunit was rapidly induced by nerve growth factor, within 60 min, in cultured adult rat dorsal root ganglion neurons. ATP synthase beta-subunit cDNA clones were isolated from a lambda library. The library was constructed using rat dorsal root ganglion mRNA that was differentially screened with cDNA-derived probes from untreated and nerve-growth-factor-treated primary cultures of adult rat dorsal root ganglion sensory neurons. Radiolabelled probes were made from submicrogram quantities of RNA, by a novel PCR-based technique, which allows small amounts of primary tissue to be used for library screening. The use of this technique in isolating novel differentially expressed mRNAs is discussed.

  1. Effects of EPA and DHA on lipid droplet accumulation and mRNA abundance of PAT proteins in caprine monocytes.

    Science.gov (United States)

    Lecchi, Cristina; Invernizzi, Guido; Agazzi, Alessandro; Modina, Silvia; Sartorelli, Paola; Savoini, Giovanni; Ceciliani, Fabrizio

    2013-04-01

    The present study investigated the in vitro effects on caprine monocytes of two ω-3 PUFAs, namely eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on lipid droplet formation, an emerging process of fundamental importance in innate immunity regulation. The mRNA abundance of PAT protein family (PLIN1, PLIN2 and PLIN3), involved in the formation and trafficking of the droplets, was also assessed. The effects of EPA and DHA on monocyte apoptosis were studied as well. The number of lipid droplets per cell was found to be dependent on both type and concentration of fatty acid. ω-3 PUFAs upregulated PLIN3 and PLIN2 gene expression, as well as apoptosis rate. The present findings suggest that PUFA might modify innate immune functions of goat monocytes by interfering with the formation of lipid droplets and by upregulating proteins belonging to PAT protein family.

  2. The impact of beta-elemene on beta-tubulin of human hepatoma hepg2 cells

    Institute of Scientific and Technical Information of China (English)

    Yuqiu Mao; Liying Ban; Jielin Zhang; Li Hou; Xiaonan Cui

    2014-01-01

    Objective:The aim of this study was to investigate the impact of beta-elemene injection on the growth and beta-tubulin of human hepatocarcinoma HepG2 cells. Methods:cellproliferation was assessed by MTT assay. cellcycle distribution was detected by flow cytometry (FCM). The mRNA expression of beta-tubulin was measured by RT-PCR. West-ern blot analysis was used to determine protein expression of beta-tubulin and the polymerization of beta-tubulin. Results:Beta-elemene injection inhibited HepG2 cells proliferation in a dose-and time-dependent manner;FCM analysis indicated beta-elemene injection induced cellcycle arrested at S phase. RT-PCR and western-blot analysis showed that beta-elemene injection down-regulated beta-tubulin expression at both mRNA and protein levels, presenting a dose-dependent manner. Moreover, beta-elemene injection reduced the polymerization of microtubules in a dose-dependent manner. Conclusion:Beta-elemene injection can inhibit the proliferation of hepatoma HepG2 cells, the mechanism might be partly related to the down-regulation of beta-tubulin and inhibition of microtubular polymerization.

  3. Accounting for experimental noise reveals that mRNA levels, amplified by post-transcriptional processes, largely determine steady-state protein levels in yeast.

    Directory of Open Access Journals (Sweden)

    Gábor Csárdi

    2015-05-01

    Full Text Available Cells respond to their environment by modulating protein levels through mRNA transcription and post-transcriptional control. Modest observed correlations between global steady-state mRNA and protein measurements have been interpreted as evidence that mRNA levels determine roughly 40% of the variation in protein levels, indicating dominant post-transcriptional effects. However, the techniques underlying these conclusions, such as correlation and regression, yield biased results when data are noisy, missing systematically, and collinear---properties of mRNA and protein measurements---which motivated us to revisit this subject. Noise-robust analyses of 24 studies of budding yeast reveal that mRNA levels explain more than 85% of the variation in steady-state protein levels. Protein levels are not proportional to mRNA levels, but rise much more rapidly. Regulation of translation suffices to explain this nonlinear effect, revealing post-transcriptional amplification of, rather than competition with, transcriptional signals. These results substantially revise widely credited models of protein-level regulation, and introduce multiple noise-aware approaches essential for proper analysis of many biological phenomena.

  4. Protein kinase C-beta II (PKC-betaII) expression in patients with colorectal cancer

    DEFF Research Database (Denmark)

    Spindler, Karen-Lise; Lindebjerg, Jan; Lahn, Michael;

    2009-01-01

    PURPOSE: Current development of targeted agents for the treatment of colorectal cancer include the clinical evaluation of kinase inhibitors, such as enzastaurin, a serine/threonine kinase inhibitor designed to suppress signaling through Protein Kinase C (PKC) and AKT pathways. Little is known abo...

  5. Ricin Inhibits Activation of the Unfolded Protein Response by Preventing Splicing of the HAC1 mRNA*

    Science.gov (United States)

    Parikh, Bijal A.; Tortora, Andrew; Li, Xiao-Ping; Tumer, Nilgun E.

    2011-01-01

    Ricin A chain (RTA) inhibits protein synthesis by removing a specific adenine from the highly conserved α-sarcin/ricin loop in the large rRNA. Expression of RTA with its own signal sequence in yeast resulted in its translocation into the endoplasmic reticulum (ER) and subsequent glycosylation. Because RTA must unfold within the ER, it may be vulnerable to host defenses, such as the unfolded protein response (UPR). UPR was induced in cells expressing an active site mutant but not the wild type RTA, indicating that the active site of RTA played a role in perturbing the ER stress response. The inactive RTA without the signal sequence did not induce UPR, indicating that translocation into the ER was critical for induction of UPR. The wild type RTA inhibited activation of UPR not only due to ER stress induced by the protein itself but also by global effectors such as tunicamycin and dithiothreitol. Mature RTA without the signal sequence also inhibited UPR, providing evidence that inhibition of UPR occurred on the cytosolic face of the ER. RTA could not inhibit UPR when the spliced form of HAC1 mRNA was provided in trans, indicating that it had a direct effect on UPR upstream of HAC1-dependent transcriptional activation. Only the precursor form of HAC1 mRNA was detected in cells expressing RTA after exposure to ER stress, demonstrating that ricin inhibits activation of UPR by preventing HAC1 mRNA splicing. The RTA mutants that depurinated ribosomes but did not kill cells were not able to inhibit activation of UPR by tunicamycin, providing evidence that the inability to activate UPR in response to ER stress contributes to the cytotoxicity of ricin. PMID:18180297

  6. Divergence of the mRNA targets for the Ssb proteins of bacteriophages T4 and RB69

    Directory of Open Access Journals (Sweden)

    Petrov Vasiliy M

    2004-09-01

    Full Text Available Abstract The single-strand binding (Ssb protein of phage T4 (T4 gp32, product of gene 32 is a mRNA-specific autogenous translational repressor, in addition to being a sequence-independent ssDNA-binding protein that participates in phage DNA replication, repair and recombination. It is not clear how this physiologically essential protein distinguishes between specific RNA and nonspecific nucleic acid targets. Here, we present phylogenetic evidence suggesting that ssDNA and specific RNA bind the same gp32 domain and that plasticity of this domain underlies its ability to configure certain RNA structures for specific binding. We have cloned and characterized gene 32 of phage RB69, a relative of T4 We observed that RB69 gp32 and T4 gp32 have nearly identical ssDNA binding domains, but diverge in their C-terminal domains. In T4 gp32, it is known that the C-terminal domain interacts with the ssDNA-binding domain and with other phage-induced proteins. In translation assays, we show that RB69 gp32 is, like T4 gp32, an autogenous translational repressor. We also show that the natural mRNA targets (translational operators for the 2 proteins are diverged in sequence from each other and yet can be repressed by either gp32. Results of chemical and RNase sensitivity assays indicate that the gp32 mRNA targets from the 2 related phages have similar structures, but differ in their patterns of contact with the 2 repressors. These and other observations suggest that a range of gp32-RNA binding specificities may evolve in nature due to plasticity of the protein-nucleic acid interaction and its response to modulation by the C-terminal domain of this translational repressor.

  7. Resveratrol Decreases TXNIP mRNA and Protein Nuclear Expressions With an Arterial Function Improvement in Old Mice.

    Science.gov (United States)

    Bedarida, Tatiana; Baron, Stephanie; Vibert, Françoise; Ayer, Audrey; Henrion, Daniel; Thioulouse, Elizabeth; Marchiol, Carmen; Beaudeux, Jean-Louis; Cottart, Charles-Henry; Nivet-Antoine, Valerie

    2016-06-01

    Aging leads to a high prevalence of glucose intolerance and cardiovascular diseases, with oxidative stress playing a potential role. Resveratrol has shown promising effects on glucose tolerance and tends to improve endothelial function in elderly patients. Thioredoxin-interacting protein (TXNIP) was recently proposed as a potential link connecting glucose metabolism to oxidative stress. Here, we investigated the resveratrol-induced improvement of arterial aging phenotype in old mice and the expression of aortic TXNIP. Using an in vivo model of old mice with or without 3-month resveratrol treatment, we investigated the effects of resveratrol on age-related impairments from a cardiovascular Doppler analysis, to a molecular level, by studying inflammation and oxidative stress factors. We found a dual effect of resveratrol, with a decrease of age-related glucose intolerance and oxidative stress imbalance leading to reduced matrix remodeling that forestalls arterial aging phenotype in terms of intima-media thickness and arterial distensibility. These results provide the first evidence that aortic TXNIP mRNA and protein nuclear expressions are increased in the arterial aging and decreased by resveratrol treatment. In conclusion, we demonstrated that resveratrol helped to restore several aging impaired processes in old mice, with a decrease of aortic TXNIP mRNA and protein nuclear expressions.

  8. Yeast mitochondrial biogenesis: a role for the PUF RNA-binding protein Puf3p in mRNA localization.

    Directory of Open Access Journals (Sweden)

    Yann Saint-Georges

    Full Text Available The asymmetric localization of mRNA plays an important role in coordinating posttranscriptional events in eukaryotic cells. We investigated the peripheral mitochondrial localization of nuclear-encoded mRNAs (MLR in various conditions in which the mRNA binding protein context and the translation efficiency were altered. We identified Puf3p, a Pumilio family RNA-binding protein, as the first trans-acting factor controlling the MLR phenomenon. This allowed the characterization of two classes of genes whose mRNAs are translated to the vicinity of mitochondria. Class I mRNAs (256 genes have a Puf3p binding motif in their 3'UTR region and many of them have their MLR properties deeply affected by PUF3 deletion. Conversely, mutations in the Puf3p binding motif alter the mitochondrial localization of BCS1 mRNA. Class II mRNAs (224 genes have no Puf3p binding site and their asymmetric localization is not affected by the absence of PUF3. In agreement with a co-translational import process, we observed that the presence of puromycin loosens the interactions between most of the MLR-mRNAs and mitochondria. Unexpectedly, cycloheximide, supposed to solidify translational complexes, turned out to destabilize a class of mRNA-mitochondria interactions. Classes I and II mRNAs, which are therefore transported to the mitochondria through different pathways, correlated with different functional modules. Indeed, Class I genes code principally for the assembly factors of respiratory chain complexes and the mitochondrial translation machinery (ribosomes and translation regulators. Class II genes encode proteins of the respiratory chain or proteins involved in metabolic pathways. Thus, MLR, which is intimately linked to translation control, and the activity of mRNA-binding proteins like Puf3p, may provide the conditions for a fine spatiotemporal control of mitochondrial protein import and mitochondrial protein complex assembly. This work therefore provides new openings

  9. Functional Significance of the Interaction between the mRNA-binding Protein, Nab2, and the Nuclear Pore-associated Protein, Mlp1, in mRNA Export* S⃞

    OpenAIRE

    Fasken, Milo B.; Stewart, Murray; Corbett, Anita H.

    2008-01-01

    Nuclear export of mRNA requires several key mRNA-binding proteins that recognize and remodel the mRNA and target it for export via interactions with the nuclear pore complex. In Saccharomyces cerevisiae, the shuttling heterogeneous nuclear ribonucleoprotein, Nab2, which is essential for mRNA export, specifically recognizes poly(A) RNA and binds to the nuclear pore-associated protein, myosin-like protein 1 (Mlp1), which functions in mRNA export and quality control. Specifically, the N-terminal...

  10. Expressions of MDM2, Livin and Caspase-3 protein and mRNA in endometrial adenocarcinomas%学位论文摘要

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    Objective To investigate the relationship of the expression of MDM2,Livin and Caspase-3 protein and mRNA in the development of endometrioid adenocarcinoma (EA). Methods The expression levels of MDM2, Livin and Caspase-3 proteins and mRNA in EA tissues (n = 72), endometrial hyperplasia tissues (n = 60) and normal tissues ( n = 30) were examined by tissue microarray technique, immunohistochemistry( SP method) and in situ hybridization method. Results The positive expression rates of MDM2, Livin and Caspase-3 protein and mRNA in EA were respectively 80. 6% ( 58/72 ), 80. 6% ( 58/72 ), 33.3% ( 24/72 ) and 73.6% ( 53/72 ), 75.0% ( 54/72 ),27.8% (20/72). The positive rates of both MDM2 and Livin protein and mRNA in EA were higher than that in normal endometrium and endometrial hyperplasia( P < 0. 01 ). However, the positive rate of Caspase-3 in EA was lower than that in normal endometrium and endometrial hyperplasia( P < 0. 01 ). The positive expressions of MDM2 protein and mRNA were not related to the histological grade, FIGO stage, depth of invasion and lymph node metastasis. The positive expressions of Livin and Caspase-3 protein and mRNA were related to histological grade (P <0. 01 ,P <0.05 ), but they were not related to FIGO stage, depth of invasion and the lymph node metastasis. The expressions of MDM2, Livin and Caspase-3 protein were positively correlated with their mRNA. The expression of Livin was negatively correlated Caspase-3. Conclusion The expressions of MDM2, Livin and Caspase-3 protein and mRNA correlate with the dedvelopment and progression of EA, which may be valuable biomarkers to detect the early carcinogenesis and prognosis of EA.

  11. Unaltered mRNA expression of calcitonin-like receptor and receptor activity modifying proteins in human arteries in stroke and myocardial infarction

    DEFF Research Database (Denmark)

    Eskesen, Karen; János, Tajti; Tibor, Hortobágyi;

    2007-01-01

    -dependent change in total RNA and level of mRNA for p-actin or GAPDH could be detected in vessels removed from 1 and 5 days post mortem. The expression of beta-actin appears lower in coronary artery than in pulmonary artery and middle cerebral artery with no significant difference for GAPDH; both worked well......-R and RAMPs in arteries from patients with hemorrhagic stroke, arteriosclerosis and acute myocardial infarction when compared to patients without these diagnoses. Thus the mRNA expression seems to be unaltered in these disorders....

  12. Association of coatomer proteins with the beta-receptor for platelet-derived growth factor

    DEFF Research Database (Denmark)

    Hansen, Klaus; Rönnstrand, L; Rorsman, C

    1997-01-01

    of intracellular vesicle transport. In order to explore the functional significance of the interaction between alpha- and beta'-COP and the PDGF receptor, a receptor mutant was made in which the conserved histidine residue 928 was mutated to an alanine residue. The mutant receptor, which was unable to bind alpha......The nonreceptor tyrosine kinase Src binds to and is activated by the beta-receptor for platelet-derived growth factor (PDGF). The interaction leads to Src phosphorylation of Tyr934 in the kinase domain of the receptor. In the course of the functional characterization of this phosphorylation, we...... noticed that components of 136 and 97 kDa bound to a peptide from this region of the receptor in a phosphorylation-independent manner. These components have now been purified and identified as alpha- and beta'-coatomer proteins (COPs), respectively. COPs are a family of proteins involved in the regulation...

  13. A poly(A) binding protein-specific sequence motif: MRTENGKSKGFGFVC binding to mRNA poly(A) and polynucleotides and its role on mRNA translation.

    Science.gov (United States)

    Rubin, H N; Halim, M N; Leavis, P C

    1994-06-01

    A consensus sequence (GKSKGFGFV) was recognized in all the sequenced poly(A) binding proteins. We synthesized a 15-amino acid peptide (corresponding to 354-368 in the yeast poly(A) binding protein) which includes the consensus sequence to test its binding affinity to different nucleotides, polynucleotides and mRNA with or without a poly(A) tail. Biochemical and biophysical studies revealed that the 15-amino acid peptide has a strong binding affinity to poly(A) alone or poly(A) attached at the 3' end of mRNA. Circular dichroism spectroscopy demonstrated that the secondary structure of the 15-mer is consistent with that expected based on the structure of the native RNP domain. Furthermore, among the various mononucleotides performed in the present studies, ATP was preferentially found to bind to the 15-mer. To further examine the biological significance of the binding of the 15-mer to the poly(A) tail of mRNA, in vitro translation of the mRNA poly(A)+ in the presence of the 15-mer drastically increased globin synthesis by almost 2-fold, while translation of the deadenylated mRNA in the presence of the 15-mer almost did not alter the rate of incorporation of radiolabeled leucine into globin.

  14. Key residues for the oligomerization of A{beta}42 protein in Alzheimer's disease

    Energy Technology Data Exchange (ETDEWEB)

    Ngo, Sam [Department of Neurology, Brain Research Institute, Molecular Biology Institute, University of California, Los Angeles, CA 90095 (United States); Guo, Zhefeng, E-mail: zhefeng@ucla.edu [Department of Neurology, Brain Research Institute, Molecular Biology Institute, University of California, Los Angeles, CA 90095 (United States)

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer A{beta} oligomers are neurotoxins and likely the causing agents for Alzheimer's disease. Black-Right-Pointing-Pointer A{beta}42 fusion protein form globular oligomers. Black-Right-Pointing-Pointer A{beta}42 fusion protein oligomers contain SDS-resistant tetramers and hexamers. Black-Right-Pointing-Pointer Cysteine substitutions at residues 31, 32, 34, 39-41 disrupt A{beta}42 oligomerization. -- Abstract: Deposition of amyloid fibrils consisting of amyloid {beta} (A{beta}) protein as senile plaques in the brain is a pathological hallmark of Alzheimer's disease. However, a growing body of evidence shows that soluble A{beta} oligomers correlate better with dementia than fibrils, suggesting that A{beta} oligomers may be the primary toxic species. The structure and oligomerization mechanism of these A{beta} oligomers are crucial for developing effective therapeutics. Here we investigated the oligomerization of A{beta}42 in the context of a fusion protein containing GroES and ubiquitin fused to the N-terminus of A{beta} sequence. The presence of fusion protein partners, in combination with a denaturing buffer containing 8 M urea at pH 10, is unfavorable for A{beta}42 aggregation, thus allowing only the most stable structures to be observed. Transmission electron microscopy showed that A{beta}42 fusion protein formed globular oligomers, which bound weakly to thioflavin T and Congo red. SDS-PAGE shows that A{beta}42 fusion protein formed SDS-resistant hexamers and tetramers. In contrast, A{beta}40 fusion protein remained as monomers on SDS gel, suggesting that the oligomerization of A{beta}42 fusion protein is not due to the fusion protein partners. Cysteine scanning mutagenesis at 22 residue positions further revealed that single cysteine substitutions of the C-terminal hydrophobic residues (I31, I32, L34, V39, V40, and I41) led to disruption of hexamer and tetramer formation, suggesting that hydrophobic interactions

  15. Factors contributing to decreased protein stability when aspartic acid residues are in {beta}-sheet regions.

    Energy Technology Data Exchange (ETDEWEB)

    Pokkuluri, P. R.; Cai, X.; Raffen, R.; Gu, M.; Stevens, F. J.; Schiffer, M.

    2002-07-01

    Asp residues are significantly under represented in {beta}-sheet regions of proteins, especially in the middle of {beta}-strands, as found by a number of studies using statistical, modeling, or experimental methods. To further understand the reasons for this under representation of Asp, we prepared and analyzed mutants of a {beta}-domain. Two Gln residues of the immunoglobulin light-chain variable domain (V{sub L}) of protein Len were replaced with Asp, and then the effects of these changes on protein stability and protein structure were studied. The replacement of Q38D, located at the end of a {beta}-strand, and that of Q89D, located in the middle of a {beta}-strand, reduced the stability of the parent immunoglobulin VL domain by 2.0 kcal/mol and 5.3 kcal/mol, respectively. Because the Q89D mutant of the wild-type V{sub L}-Len domain was too unstable to be expressed as a soluble protein, we prepared the Q89D mutant in a triple mutant background, V{sub L}-Len M4L/Y27dD/T94H, which was 4.2 kcal/mol more stable than the wild-type V{sub L}-Len domain. The structures of mutants V{sub L}-Len Q38D and V{sub L}-Len Q89D/M4L/Y27dD/T94H were determined by X-ray diffraction at 1.6 A resolution. We found no major perturbances in the structures of these QD mutant proteins relative to structures of the parent proteins. The observed stability changes have to be accounted for by cumulative effects of the following several factors: (1) by changes in main-chain dihedral angles and in side-chain rotomers, (2) by close contacts between some atoms, and, most significantly, (3) by the unfavorable electrostatic interactions between the Asp side chain and the carbonyls of the main chain. We show that the Asn side chain, which is of similar size but neutral, is less destabilizing. The detrimental effect of Asp within a {beta}-sheet of an immunoglobulin-type domain can have very serious consequences. A somatic mutation of a {beta}-strand residue to Asp could prevent the expression of the

  16. Topological switching between an alpha-beta parallel protein and a remarkably helical molten globule.

    Science.gov (United States)

    Nabuurs, Sanne M; Westphal, Adrie H; aan den Toorn, Marije; Lindhoud, Simon; van Mierlo, Carlo P M

    2009-06-17

    Partially folded protein species transiently exist during folding of most proteins. Often these species are molten globules, which may be on- or off-pathway to native protein. Molten globules have a substantial amount of secondary structure but lack virtually all the tertiary side-chain packing characteristic of natively folded proteins. These ensembles of interconverting conformers are prone to aggregation and potentially play a role in numerous devastating pathologies, and thus attract considerable attention. The molten globule that is observed during folding of apoflavodoxin from Azotobacter vinelandii is off-pathway, as it has to unfold before native protein can be formed. Here we report that this species can be trapped under nativelike conditions by substituting amino acid residue F44 by Y44, allowing spectroscopic characterization of its conformation. Whereas native apoflavodoxin contains a parallel beta-sheet surrounded by alpha-helices (i.e., the flavodoxin-like or alpha-beta parallel topology), it is shown that the molten globule has a totally different topology: it is helical and contains no beta-sheet. The presence of this remarkably nonnative species shows that single polypeptide sequences can code for distinct folds that swap upon changing conditions. Topological switching between unrelated protein structures is likely a general phenomenon in the protein structure universe.

  17. Affibody-beta-galactosidase immunoconjugates produced as soluble fusion proteins in the Escherichia coli cytosol.

    Science.gov (United States)

    Rönnmark, Jenny; Kampf, Caroline; Asplund, Anna; Höidén-Guthenberg, Ingmarie; Wester, Kenneth; Pontén, Fredrik; Uhlén, Mathias; Nygren, Per-Ake

    2003-10-01

    Recombinant immunoconjugates constitute a novel class of immunoassay reagents produced by genetic fusion between an antigen recognizing moiety and a reporter enzyme or fluorescent protein, obviating the need for chemical coupling. In this work, we describe the construction, Escherichia coli production and characterization of recombinant beta-galactosidase (beta-gal)-based immunoconjugates directed to human immunoglobulin A (IgA). As the antigen recognizing moieties, either monovalent or dimeric (head-to-tail) versions of an IgA-specific affibody (Z(IgA1)) were used, previously selected in vitro from a protein library based on combinatorial engineering of a single staphylococcal protein A domain. To increase the likelihood of proper presentation on the assembled homotetrameric enzyme surface, the affibody moieties were linked to the N-terminus of the enzyme subunits via a heptapeptide linker sequence. The two resulting immunoconjugates Z(IgA1)-beta-gal and (Z(IgA1))(2)-beta-gal, containing four and eight affibody moieties per enzyme, respectively, could be expressed as soluble and proteolytically stable proteins intracellularly in E. coli from where they were purified to high purity by a single anion exchange chromatography step. The yields of immunoconjugates were in the range 200-400 mg/l culture. Biosensor-binding studies showed that both the Z(IgA1)-beta-gal and (Z(IgA1))(2)-beta-gal immunoconjugates were capable of selective IgA-recognition, but with an apparent higher binding affinity for the variant containing divalent affibody moieties, presumably due to avidity effects. The applicability of this class of recombinant immunoconjugates was demonstrated by IgA detection in enzyme-linked immunosorbent assay (ELISA) and dot-blot analyses. In addition, using human kidney biopsy samples from a nephropathy patient, IgA depositions in glomeruli could be detected by immunohistochemistry with low background staining of tissue.

  18. Ranking beta sheet topologies with applications to protein structure prediction

    DEFF Research Database (Denmark)

    Fonseca, Rasmus; Helles, Glennie; Winter, Pawel

    2011-01-01

    One reason why ab initio protein structure predictors do not perform very well is their inability to reliably identify long-range interactions between amino acids. To achieve reliable long-range interactions, all potential pairings of ß-strands (ß-topologies) of a given protein are enumerated......, including the native ß-topology. Two very different ß-topology scoring methods from the literature are then used to rank all potential ß-topologies. This has not previously been attempted for any scoring method. The main result of this paper is a justification that one of the scoring methods, in particular...... of this paper is a method to deal with the inaccuracies of secondary structure predictors when enumerating potential ß-topologies. The results reported in this paper are highly relevant for ab initio protein structure prediction methods based on decoy generation. They indicate that decoy generation can...

  19. Microglia, neuroinflammation, and beta-amyloid protein in Alzheimer's disease.

    Science.gov (United States)

    Cai, Zhiyou; Hussain, M Delwar; Yan, Liang-Jun

    2014-05-01

    Compelling evidence from basic molecular biology has demonstrated the dual roles of microglia in the pathogenesis of Alzheimer's disease (AD). On one hand, microglia are involved in AD pathogenesis by releasing inflammatory mediators such as inflammatory cytokines, complement components, chemokines, and free radicals that are all known to contribute to beta-amyloid (Aβ) production and accumulation. On the other hand, microglia are also known to play a beneficial role in generating anti-Aβ antibodies and stimulating clearance of amyloid plaques. Aβ itself, an inducer of microglia activation and neuroinflammation, has been considered as an underlying and unifying factor in the development of AD. A vicious cycle of inflammation has been formed between Aβ accumulation, activated microglia, and microglial inflammatory mediators, which enhance Aβ deposition and neuroinflammation. Thus, inhibiting the vicious cycle seems to be a promising treatment to restrain further development of AD. With increasing research efforts on microglia in AD, intervention of microglia activation and neuroinflammation in AD may provide a potential target for AD therapy in spite of the provisional failure of nonsteroidal antiinflammatory drugs in clinical trials.

  20. Poultry fat decreased fatty acid transporter protein mRNA expression and affected fatty acid composition in chickens

    Directory of Open Access Journals (Sweden)

    Yuan Jianmin

    2012-05-01

    Full Text Available Abstract Background A study was undertaken to examine the effects of poultry fat (PF compared with those of soybean oil (SBO on intestinal development, fatty acid transporter protein (FATP mRNA expression, and fatty acid composition in broiler chickens. A total of 144 day-old male commercial broilers were randomly allocated to 2 treatment groups (6 replicates of 12 chicks for each treatment and fed isocaloric diets containing 3.0% PF or 2.7% SBO at 0 to 3 wk and 3.8% PF or 3.5% SBO at 4 to 6 wk, respectively. Results PF had no influence on intestinal morphology, weight, or DNA, RNA, or protein concentrations at 2, 4, and 6 wk of age. However, compared with SBO, PF significantly decreased FATP mRNA abundance at 4 wk (P = 0.009 and 6 wk of age (P P = 0.039; and decreased C18:2 (P = 0.015, C18:3 (P P = 0.018, Σ-polyunsaturated fatty acids (Σ-PUFA (P = 0.020, and the proportion of PUFA (P P = 0.010, C18:3 (P P P = 0.005, and the proportion of PUFA (P  Conclusions PF decreases FATP and L-FABP mRNA expression and decreased the proportion of PUFA in the intestinal mucosa and breast muscle.

  1. Structure of the gene encoding the murine protein kinase CK2 beta subunit

    DEFF Research Database (Denmark)

    Boldyreff, B; Issinger, O G

    1995-01-01

    The mouse protein kinase CK2 beta subunit gene (Csnk2b) is composed of seven exons contained within 7874 bp. The exon and intron lengths extend from 76 to 321 and 111 to 1272 bp, respectively. The lengths of the murine coding exons correspond exactly to the lengths of the exons in the human CK2...

  2. Rat beta(3)-adrenoceptor protein expression : antibody validation and distribution in rat gastrointestinal and urogenital tissues

    NARCIS (Netherlands)

    Cernecka, Hana; Pradidarcheep, Wisuit; Lamers, Wouter H.; Schmidt, Martina; Michel, Martin C.

    2014-01-01

    beta(3)-Adrenoceptors play important roles in the regulation of urogenital and probably gastrointestinal function. However, despite recent progress, their detection at the protein level has remained difficult due to a lack of sufficiently validated selective antibodies. Therefore, we have explored t

  3. Structure of protein kinase CK2: dimerization of the human beta-subunit

    DEFF Research Database (Denmark)

    Boldyreff, B; Mietens, U; Issinger, O G

    1996-01-01

    Protein kinase CK2 has been shown to be elevated in all so far investigated solid tumors and its catalytic subunit has been shown to serve as an oncogene product. CK2 is a heterotetrameric serine-threonine kinase composed of two catalytic (alpha and/or alpha') and two regulatory beta...

  4. Ability of CK2beta to selectively regulate cellular protein kinases

    DEFF Research Database (Denmark)

    Olsen, Birgitte; Guerra, Barbara

    2008-01-01

    The Wee1 protein kinase plays a prominent role in keeping cyclin dependent kinase 1 (CDK1) inactive during the G2 phase of the cell cycle. At the onset of mitosis, Wee1 is ubiquitinated by the E3 ubiquitin ligase SCF(beta-TrCP) and subsequently degraded by the proteasome machinery. Previously, it...

  5. Protein and fat mobilization and associations with serum beta-hydroxybutyrate concentrations in dairy cows

    NARCIS (Netherlands)

    van der Drift, S. G. A.; Houweling, M.; Schonewille, J. T.; Tielens, A. G. M.; Jorritsma, R.

    2012-01-01

    The objective of this study was to obtain information on variation between dairy cows in muscle and fat tissue mobilization around parturition and to study the association between protein and fat mobilization and serum beta-hydroxybutyrate (BHBA) concentrations (hyperketonemia) in this period. Thirt

  6. Symmetric key structural residues in symmetric proteins with beta-trefoil fold.

    Directory of Open Access Journals (Sweden)

    Jianhui Feng

    Full Text Available To understand how symmetric structures of many proteins are formed from asymmetric sequences, the proteins with two repeated beta-trefoil domains in Plant Cytotoxin B-chain family and all presently known beta-trefoil proteins are analyzed by structure-based multi-sequence alignments. The results show that all these proteins have similar key structural residues that are distributed symmetrically in their structures. These symmetric key structural residues are further analyzed in terms of inter-residues interaction numbers and B-factors. It is found that they can be distinguished from other residues and have significant propensities for structural framework. This indicates that these key structural residues may conduct the formation of symmetric structures although the sequences are asymmetric.

  7. P-cadherin and beta-catenin are useful prognostic markers in breast cancer patients; beta-catenin interacts with heat shock protein Hsp27.

    Science.gov (United States)

    Fanelli, Mariel A; Montt-Guevara, Magdalena; Diblasi, Angela M; Gago, Francisco E; Tello, Olga; Cuello-Carrión, F Darío; Callegari, Eduardo; Bausero, Maria A; Ciocca, Daniel R

    2008-01-01

    The cadherin-catenin proteins have in common with heat shock proteins (HSP) the capacity to bind/interact proteins of other classes. Moreover, there are common molecular pathways that connect the HSP response and the cadherin-catenin protein system. In the present study, we have explored whether in breast cancer the HSP might interact functionally with the cadherin-catenin cell adhesion system. Beta-catenin was immunoprecipitated from breast cancer biopsy samples, and the protein complexes isolated in this way were probed with antibodies against HSP family members. We are thus the first to demonstrate a specific interaction between beta-catenin and Hsp27. However, beta-catenin did not bind Hsp60, Hsp70, Hsp90, gp96, or the endoplasmic reticulum stress response protein CHOP. To confirm the finding of Hsp27-beta-catenin interaction, the 27-kDa immunoprecipitated band was excised from one-dimensional polyacrylamide gel electrophoresis gels and submitted to liquid chromatography-tandem mass spectrometry with electrospray ionization, confirming a role for Hsp27. In addition, beta-catenin interacted with other proteins including heat shock transcription factor 1, P-cadherin, and caveolin-1. In human breast cancer biopsy samples, beta-catenin was coexpressed in the same tumor areas and in the same tumor cells that expressed Hsp27. However, this coexpression was strong when beta-catenin was present in the cytoplasm of the tumor cells and not when beta-catenin was expressed at the cell surface only. Furthermore, murine breast cancer cells transfected with hsp25 showed a redistribution of beta-catenin from the cell membrane to the cytoplasm. When the prognostic significance of cadherin-catenin expression was examined by immunohistochemistry in breast cancer patients (n = 215, follow-up = >10 years), we found that the disease-free survival and overall survival were significantly shorter for patients expressing P-cadherin and for patients showing expression of beta-catenin in

  8. Translational Control Protein 80 Stimulates IRES-Mediated Translation of p53 mRNA in Response to DNA Damage

    Directory of Open Access Journals (Sweden)

    Marie-Jo Halaby

    2015-01-01

    Full Text Available Synthesis of the p53 tumor suppressor increases following DNA damage. This increase and subsequent activation of p53 are essential for the protection of normal cells against tumorigenesis. We previously discovered an internal ribosome entry site (IRES that is located at the 5′-untranslated region (UTR of p53 mRNA and found that the IRES activity increases following DNA damage. However, the mechanism underlying IRES-mediated p53 translation in response to DNA damage is still poorly understood. In this study, we discovered that translational control protein 80 (TCP80 has increased binding to the p53 mRNA in vivo following DNA damage. Overexpression of TCP80 also leads to increased p53 IRES activity in response to DNA damage. TCP80 has increased association with RNA helicase A (RHA following DNA damage and overexpression of TCP80, along with RHA, leads to enhanced expression of p53. Moreover, we found that MCF-7 breast cancer cells with decreased expression of TCP80 and RHA exhibit defective p53 induction following DNA damage and diminished expression of its downstream target PUMA, a proapoptotic protein. Taken together, our discovery of the function of TCP80 and RHA in regulating p53 IRES and p53 induction following DNA damage provides a better understanding of the mechanisms that regulate IRES-mediated p53 translation in response to genotoxic stress.

  9. Pokeweed antiviral protein restores levels of cellular APOBEC3G during HIV-1 infection by depurinating Vif mRNA.

    Science.gov (United States)

    Krivdova, Gabriela; Hudak, Katalin A

    2015-10-01

    Pokeweed antiviral protein (PAP) is an RNA glycosidase that inhibits production of human immunodeficiency virus type 1 (HIV-1) when expressed in human culture cells. Previously, we showed that the expression of PAP reduced the levels of several viral proteins, including virion infectivity factor (Vif). However, the mechanism causing Vif reduction and the consequences of the inhibition were not determined. Here we show that the Vif mRNA is directly depurinated by PAP. Because of depurination at two specific sites within the Vif ORF, Vif levels decrease during infections and the progeny viruses that are generated are ∼ 10-fold less infectious and compromised for proviral integration. These results are consistent with PAP activity inhibiting translation of Vif, which in turn reduces the effect of Vif to inactivate the host restriction factor APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like editing complex 3G). Our findings identify Vif mRNA as a new substrate for PAP and demonstrate that derepression of innate immunity against HIV-1 contributes to its antiviral activity.

  10. hA molecular switch in amyloid assembly: Met35 and amyloid beta-protein oligomerization

    OpenAIRE

    Bitan, Gal; Tarus, Bogdan; Vollers, Sabrina S.; Lashuel, Hilal A.; Condron, Margaret M.; Straub, John E.; Teplow, David B.

    2003-01-01

    Aberrant protein oligomerization is an important pathogenetic process in vivo. In Alzheimer's disease (AD), the amyloid beta-protein (Abeta) forms neurotoxic oligomers. The predominant in vivo Abeta alloforms, Abeta40 and Abeta42, have distinct oligomerization pathways. Abeta42 monomers oligomerize into pentamer/hexamer units (paranuclei) which self-associate to form larger oligomers. Abeta40 does not form these paranuclei, a fact which may explain the particularly strong linkage of Abeta42 w...

  11. Bed rest reduces metabolic protein content and abolishes exercise-induced mRNA responses in human skeletal muscle

    DEFF Research Database (Denmark)

    Jørgensen, Stine Ringholm; Biensø, Rasmus S; Kiilerich, Kristian

    2011-01-01

    Background: The aim was to test the hypothesis that one week of bed rest will reduce mitochondrial number and expression and activity of oxidative proteins in human skeletal muscle, but that exercise-induced intracellular signaling as well as mRNA and microRNA (miR) responses are maintained after......-legged knee extensor exercise performed before and after bed rest. Results: Maximal oxygen uptake decreased 5% and exercise endurance decreased non-significantly 25% by bed rest. Bed rest reduced skeletal muscle mitochondrial DNA/nuclear DNA content 15%, hexokinase II and sirtuin 1 protein content ~45%, 3...... bed rest. Research Design and Methods: Twelve young, healthy, male subjects completed 7 days of bed rest with vastus lateralis muscle biopsies taken before and after bed rest. In addition, muscle biopsies were obtained from 6 of the subjects prior to, immediately after and 3h after 45 min one...

  12. Structural Aspects for Evolution of [beta]-Lactamases from Penicillin-Binding Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Meroueh, Samy O.; Minasov, George; Lee, Wenlin; Shoichet, Brian K.; Mobashery, Shahriar (NWU); (UCSF); (Notre)

    2010-03-08

    Penicillin-binding proteins (PBPs), biosynthetic enzymes of bacterial cell wall assembly, and {beta}-lactamases, resistance enzymes to {beta}-lactam antibiotics, are related to each other from an evolutionary point of view. Massova and Mobashery (Antimicrob. Agents Chemother. 1998, 42, 1-17) have proposed that for {beta}-lactamases to have become effective at their function as antibiotic resistance enzymes, they would have had to undergo structure alterations such that they would not interact with the peptidoglycan, which is the substrate for PBPs. A cephalosporin analogue, 7{beta}-[N-Acetyl-L-alanyl-{gamma}-D-glutamyl-L-lysine]-3-acetoxymethyl-3-cephem-carboxylic acid (compound 6), was conceived and synthesized to test this notion. The X-ray structure of the complex of this cephalosporin bound to the active site of the deacylation-deficient Q120L/Y150E variant of the class C AmpC {beta}-lactamase from Escherichia coli was solved at 1.71 {angstrom} resolution. This complex revealed that the surface for interaction with the strand of peptidoglycan that acylates the active site, which is present in PBPs, is absent in the {beta}-lactamase active site. Furthermore, insertion of a peptide in the {beta}-lactamase active site at a location where the second strand of peptidoglycan in some PBPs binds has effectively abolished the possibility for such interaction with the {beta}-lactamase. A 2.6 ns dynamics simulation was carried out for the complex, which revealed that the peptidoglycan surrogate (i.e., the active-site-bound ligand) undergoes substantial motion and is not stabilized for binding within the active site. These factors taken together disclose the set of structure modifications in the antibiotic resistance enzyme that prevent it from interacting with the peptidoglycan, en route to achieving catalytic proficiency for their intended function.

  13. Progesterone Downregulates Oestrogen-Induced Expression of CFTR and SLC26A6 Proteins and mRNA in Rats’ Uteri

    Directory of Open Access Journals (Sweden)

    K. Gholami

    2012-01-01

    Full Text Available Under progesterone (P dominance, fluid loss assists uterine closure which is associated with pH reduction. We hypothesize that P inhibits uterine fluid secretion and HCO3- transport. Aim. to investigate the expression of Cystic Fibrosis Transmembrane Regulator (CFTR and Cl−/HCO3- exchanger (SLC26A6 under P effect. Method. Uteri from ovariectomized steroid replaced and intact rats at different stages of oestrous cycle were analyzed for changes in protein and mRNA expressions. Results. P inhibits CFTR and SLC26A6 proteins and mRNA expression while oestrogen (E causes vice versa. E treatment followed by P causes a reduction in these transporters’ mRNA and protein. Similar changes occur throughout the oestrous cycle; that is, CFTR mRNA expression was high at proestrus while SLC26A6 mRNA and protein expressions were increased at proestrus and estrus. At diestrus, however, the expression of these transporters’ protein and mRNA was reduced. Conclusion. Inhibition of CFTR and SLC26A6 expressions may explain the reduced fluid volume and pH under P-mediated effect.

  14. Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

    Science.gov (United States)

    Kallehauge, Thomas Beuchert; Li, Shangzhong; Pedersen, Lasse Ebdrup; Ha, Tae Kwang; Ley, Daniel; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup; Lee, Gyun Min; Lewis, Nathan E.

    2017-01-01

    Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we identified the unnecessary resistance marker NeoR to be a highly transcribed and translated gene. Through siRNA knock-down of NeoR, we improved the production- and growth capacity of the host cell. Thus, ribosomal profiling provides valuable insights into translation in CHO cells and can guide efforts to enhance protein production. PMID:28091612

  15. Protein identification and mRNA analysis of phyto- chrome-regulated genes in Arabidopsis under red light

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Phytochromes are a family of plant photoreceptors that mediate physiological and developmental re- sponses to red and far-red light. According to the affymetrix ATH1 microarray, phytochrome A (phyA) and phytochrome B (phyB) together play a key role in transducing the Rc signals to light-responsive genes. In order to select those red light-responsive genes associated with phyA or phyB, a proteomic approach based on two-dimensional gel electrophoresis (2-DE) was used to compare the protein ex- pression patterns of the phyAphyB double mutant and the wild type of Arabidopsis thaliana (col-4) which grew under constant red light conditions for 7 d. Thirty-two protein spots which exhibited dif- ferences in protein abundance were identified by matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry. The expression of ten genes corresponding to ten protein spots was analyzed by a semiquantitative reverse transcription-polymerase chain reaction. Two of the ten genes were confirmed by quantitative PCR (Q-PCR). The results showed that phytochromes may exert their function by regulating mRNA or protein expressions. Proteomic analysis may provide a novel pathway for identifying phytochrome-dependent genes.

  16. Protein identification and mRNA analysis of phyto-chrome-regulated genes in Arabidopsis under red light

    Institute of Scientific and Technical Information of China (English)

    LI Xu; YANG YueJun; LI Yan; WANG Jie; XIAO XiaoJuan; GUO XinHong; TANG DongYing; LIU XuanMing

    2009-01-01

    Phytochromes are a family of plant photoreceptors that mediate physiological and developmental re-sponses to red and far-red light. According to the affymetrix ATH1 microarray, phytochrome A (phyA)and phytochrome B (phyB) together play a key role in transducing the Rc signals to light-responsive genes. In order to select those red light-responsive genes associated with phyA or phyB, a proteomic approach based on two-dimensional gel electrophoresis (2-DE) was used to compare the protein ex-pression patterns of the phyAphyB double mutant and the wild type of Arabidopsis thaliana (col-4)which grew under constant red light conditions for 7 d. Thirty-two protein spots which exhibited dif-ferences in protein abundance were identified by matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry. The expression of ten genes corresponding to ten protein spots was analyzed by a semiquantitative reverse transcription-polymerase chain reaction. Two of the ten genes were confirmed by quantitative PCR (Q-PCR). The results showed that phytochromes may exert their function by regulating mRNA or protein expressions. Proteomic analysis may provide a novel pathway for identifying phytochrome-dependent genes.

  17. Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

    Science.gov (United States)

    Kallehauge, Thomas Beuchert; Li, Shangzhong; Pedersen, Lasse Ebdrup; Ha, Tae Kwang; Ley, Daniel; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup; Lee, Gyun Min; Lewis, Nathan E.

    2017-01-01

    Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we identified the unnecessary resistance marker NeoR to be a highly transcribed and translated gene. Through siRNA knock-down of NeoR, we improved the production- and growth capacity of the host cell. Thus, ribosomal profiling provides valuable insights into translation in CHO cells and can guide efforts to enhance protein production.

  18. Gallium nitrate increases type I collagen and fibronectin mRNA and collagen protein levels in bone and fibroblast cells.

    Science.gov (United States)

    Bockman, R S; Guidon, P T; Pan, L C; Salvatori, R; Kawaguchi, A

    1993-08-01

    Gallium is a Group IIIa transitional element with therapeutic efficacy in the treatment of metabolic bone disorders. Previously described antiresorptive effects of gallium on osteoclasts are not sufficient to account for the full range of effects of gallium on bone structure and metabolism. We have recently shown that gallium nitrate inhibits osteocalcin gene expression and the synthesis of osteocalcin protein, an osteoblast-specific bone matrix protein that is thought to serve as a signal to trigger osteoclastic resorption. Here we present evidence for an additional mechanism by which gallium may function to augment bone mass by altering matrix protein synthesis by osteoblastic and fibroblastic cells. Rat calvarial explants exposed to gallium nitrate for 48 h showed increased incorporation of 3H-proline into hydroxyproline and collagenase digestible protein. In addition, gallium treatment increased steady-state mRNA levels for fibronectin and type I procollagen chains in primary rat calvarial osteoblast-enriched cultures, the ROS 17/2.8 osteoblastic osteosarcoma line, and nontransformed human dermal fibroblasts. These findings suggest that the exposure of mesenchymally-derived cells to gallium results in an altered pattern of matrix protein synthesis that would favor increased bone formation.

  19. Effects of red orpiment on cell morphology and expression of PML mRNA and protein in NB4 and HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    钟璐; 陈芳源; 韩洁英; 邵念贤; 欧阳仁荣

    2003-01-01

    Objective To investigate the effects of red orpiment on cell morphology, expression of promyelocytic leukemia (PML) mRNA and its protein localization in NB4 and HL-60 cell lines.Methods Cell morphology was assayed by Wright's staining and fluorescence staining, while PML mRNA expression was determined by RT-PCR. PML protein localization by evaluated by immunofluorescence staining. Results The typical apoptosis was found in NB4 and HL-60 cells after treatment with red orpiment. The fusion protein was no longer observed in NB4 cells, PML protein was relocated, and then degraded. In HL-60 cells, PML protein underwent a similar progress. The expression of promyelocytic leukemia (PML) mRNA was not changed in the treated cells.Conclusion Red orpiment inhibits the proliferation of leukemia cells by inducing them to undergo apoptosis.

  20. Receptor tyrosine phosphatase beta is expressed in the form of proteoglycan and binds to the extracellular matrix protein tenascin

    DEFF Research Database (Denmark)

    Barnea, G; Grumet, M; Milev, P;

    1994-01-01

    The extracellular domain of receptor type protein tyrosine phosphatase beta (RPTP beta) exhibits striking sequence similarity with a soluble, rat brain chondroitin sulfate proteoglycan (3F8 PG). Immunoprecipitation experiments of cells transfected with RPTP beta expression vector and metabolically...... labeled with [35S]sulfate and [35S]methionine indicate that the transmembrane form of RPTP beta is indeed a chondroitin sulfate proteoglycan. The 3F8 PG is therefore a variant form composed of the entire extracellular domain of RPTP beta probably generated by alternative RNA splicing. Previous...

  1. Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3 overexpression in pancreatic ductal adenocarcinoma correlates with poor survival

    Directory of Open Access Journals (Sweden)

    Scudamore Charles H

    2010-02-01

    Full Text Available Abstract Background Pancreatic ductal adenocarcinoma is a lethal disease with a 5-year survival rate of 4% and typically presents in an advanced stage. In this setting, prognostic markers identifying the more agrressive tumors could aid in managment decisions. Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3, also known as IMP3 or KOC is an oncofetal RNA-binding protein that regulates targets such as insulin-like growth factor-2 (IGF-2 and ACTB (beta-actin. Methods We evaluated the expression of IGF2BP3 by immunohistochemistry using a tissue microarray of 127 pancreatic ductal adenocarcinomas with tumor grade 1, 2 and 3 according to WHO criteria, and the prognostic value of IGF2BP3 expression. Results IGF2BP3 was found to be selectively overexpressed in pancreatic ductal adenocarcinoma tissues but not in benign pancreatic tissues. Nine (38% patient samples of tumor grade 1 (n = 24 and 27 (44% of tumor grade 2 (n = 61 showed expression of IGF2BP3. The highest rate of expression was seen in poorly differentiated specimen (grade 3, n = 42 with 26 (62% positive samples. Overall survival was found to be significantly shorter in patients with IGF2BP3 expressing tumors (P = 0.024; RR 2.3, 95% CI 1.2-4.8. Conclusions Our data suggest that IGF2BP3 overexpression identifies a subset of pancreatic ductal adenocarcinomas with an extremely poor outcome and supports the rationale for developing therapies to target the IGF pathway in this cancer.

  2. Intracellular localization and interaction of mRNA binding proteins as detected by FRET

    Directory of Open Access Journals (Sweden)

    Port J

    2010-09-01

    Full Text Available Abstract Background A number of RNA binding proteins (BPs bind to A+U rich elements (AREs, commonly present within 3'UTRs of highly regulated RNAs. Individual RNA-BPs proteins can modulate RNA stability, RNA localization, and/or translational efficiency. Although biochemical studies have demonstrated selectivity of ARE-BPs for individual RNAs, less certain is the in vivo composition of RNA-BP multiprotein complexes and how their composition is affected by signaling events and intracellular localization. Using FRET, we previously demonstrated that two ARE-BPs, HuR and AUF1, form stable homomeric and heteromeric associations in the nucleus and cytoplasm. In the current study, we use immuno-FRET of endogenous proteins to examine the intracellular localization and interactions of HuR and AUF1 as well as KSRP, TIA-1, and Hedls. These results were compared to those obtained with their exogenously expressed, fluorescently labeled counterparts. Results All ARE-BPs examined were found to colocalize and to form stable associations with selected other RNA-BPs in one or more cellular locations variably including the nucleus, cytoplasm (in general, or in stress granules or P bodies. Interestingly, FRET based interaction of the translational suppressor, TIA-1, and the decapping protein, Hedls, was found to occur at the interface of stress granules and P bodies, dynamic sites of intracellular RNA storage and/or turnover. To explore the physical interactions of RNA-BPs with ARE containing RNAs, in vitro transcribed Cy3-labeled RNA was transfected into cells. Interestingly, Cy3-RNA was found to coalesce in P body like punctate structures and, by FRET, was found to interact with the RNA decapping proteins, Hedls and Dcp1. Conclusions Biochemical methodologies, such as co-immunoprecipitation, and cell biological approaches such as standard confocal microscopy are useful in demonstrating the possibility of proteins and/or proteins and RNAs interacting. However, as

  3. Inhibition of beta-site amyloid precursor protein-cleaving enzyme and beta-amyloid precursor protein genes in SK-N-SH cells

    Institute of Scientific and Technical Information of China (English)

    Suqin Gao; Lin Sun; Enji Han; Hongshun Qi; Jinbo Feng; Shunliang Xu; Wen Xia

    2009-01-01

    BACKGROUND:Previous studies have demonstrated that Piper futokadsura stem selectively inhibits expression of amyloid precursor protein (APP) at the mRNA level.In addition,the piperlonguminine (A) and dihydropiperlonguminine (B) components (1:0.8),which can be separated from Futokadsura stem,selectively inhibit expression of the APP at mRNA and protein levels.OBJECTIVE:Based on previous findings,the present study investigated the effects of β-site amyloid precursor protein cleaving enzyme (BACE1) and APP genes on the production of β-amyloid peptide 42 (Aβ42) in human neuroblastoma cells (SK-N-SH cells) using small interfering RNAs (siRNAs) and A/B components separated from Futokadsura stem,respectively.DESIGN,TIME AND SETTING:A gene interference-based randomized,controlled,in vitro experiment was performed at the Key Laboratory of Cardiovascular Remodeling and Function Research,Ministries of Education and Public Health,and Institute of Pharmacologic Research,School of Pharmaceutical Science & Department of Biochemistry,School of Medicine,Shandong University between July 2006 and December 2007.MATERIALS:SK-N-SH cells were provided by Shanghai Institutes of Biological Sciences,Chinese Academy of Sciences,Shanghai,China;mouse anti-human BACE1 monoclonal antibody was purchased from R&D Systems,USA;mouse anti-human APP monoclonal antibody was purchased from Cell Signaling Technology,USA;and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG was provided by Sigma,USA.METHODS:The human BACE1 cDNA sequence was obtained from NCBI website (www.ncbi.nlm.nih.gov/sites/entrez).Three pairs of siRNAs,specific to human BACE1 gene,were synthesized through the use of Silencer? pre-designed siRNA specification,and were transfected into SK-N-SH cells with siPORT NeoFX transfection agent to compare the effects of different concentrations of siRNAs (10-50 nmol/L) on SK-N-SH cells.Futokadsura stem was separated and purified with chemical methods,and the crystal was composed of

  4. Expression of transforming growth factor beta (TGF beta) receptors and expression of TGF beta 1, TGF beta 2 and TGF beta 3 in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Damstrup, L; Rygaard, K; Spang-Thomsen, M;

    1993-01-01

    A panel of 21 small cell lung cancer cell (SCLC) lines were examined for the presence of Transforming growth factor beta receptors (TGF beta-r) and the expression of TGF beta mRNAs. By the radioreceptor assay we found high affinity receptors to be expressed in six cell lines. scatchard analysis...... of the binding data demonstrated that the cells bound between 4.5 and 27.5 fmol mg-1 protein with a KD ranging from 16 to 40 pM. TGF beta 1 binding to the receptors was confirmed by cross-linking TGF beta 1 to the TGF beta-r. Three classes of TGF beta-r were demonstrated, type I and type II receptors with M......(r) = 65,000 and 90,000 and the betaglycan (type III) with M(r) = 280,000. Northern blotting showed expression of TGF beta 1 mRNA in ten, TGF beta 2 mRNA in two and TGF beta 3 mRNA in seven cell lines. Our results provide, for the first time, evidence that a large proportion of a broad panel of SCLC cell...

  5. Association Analysis of Myosin Heavy-chain Genes mRNA Transcription with the Corresponding Proteins Expression of Muscle in Growing Pigs

    Directory of Open Access Journals (Sweden)

    X. M. Men

    2016-04-01

    Full Text Available The goal of this work was to investigate the correlations between MyHC mRNA transcription and their corresponding protein expressions in porcine longissimus muscle (LM during postnatal growth of pigs. Five DLY (Duroc×Landrace×Yorkshire crossbred pigs were selected, slaughtered and sampled at postnatal 7, 30, 60, 120, and 180 days, respectively. Each muscle was subjected to quantity MyHCs protein contents through an indirect enzyme-linked immunosorbent assay (ELISA, to quantity myosin heavy-chains (MyHCs mRNA abundances using real-time polymerase chain reaction. We calculated the proportion (% of each MyHC to total of four MyHC for two levels, respectively. Moreover, the activities of several key energy metabolism enzymes were determined in LM. The result showed that mRNA transcription and protein expression of MyHC I, IIa, IIx and IIb in LM all presented some obvious changes with postnatal aging of pigs, especially at the early stage after birth, and their mRNA transcriptions were easy to be influenced than their protein expressions. The relative proportion of each MyHC mRNA was significantly positively related to that of its corresponding protein (p<0.01, and MyHC I mRNA proportion was positively correlated with creatine kinase (CK, succinate dehydrogenase (SDH, malate dehydrogenase (MDH activities (p<0.05. These data suggested that MyHC mRNA transcription can be used to reflect MyHC expression, metabolism property and adaptive plasticity of porcine skeletal muscles, and MyHC mRNA composition could be a molecular index reflecting muscle fiber type characteristics.

  6. Role of a redox-based methylation switch in mRNA life cycle (pre- and post-transcriptional maturation) and protein turnover: implications in neurological disorders.

    Science.gov (United States)

    Trivedi, Malav S; Deth, Richard C

    2012-01-01

    Homeostatic synaptic scaling in response to neuronal stimulus or activation, and due to changes in cellular niche, is an important phenomenon for memory consolidation, retrieval, and other similar cognitive functions (Turrigiano and Nelson, 2004). Neurological disorders and cognitive disabilities in autism, Rett syndrome, schizophrenia, dementia, etc., are strongly correlated to alterations in protein expression (both synaptic and cytoplasmic; Cajigas et al., 2010). This correlation suggests that efficient temporal regulation of synaptic protein expression is important for synaptic plasticity. In addition, equilibrium between mRNA processing, protein translation, and protein turnover is a critical sensor/trigger for recording synaptic information, normal cognition, and behavior (Cajigas et al., 2010). Thus a regulatory switch, which controls the lifespan, maturation, and processing of mRNA, might influence cognition and adaptive behavior. Here, we propose a two part novel hypothesis that methylation might act as this suggested coordinating switch to critically regulate mRNA maturation at (1) the pre-transcription level, by regulating precursor-RNA processing into mRNA, via other non-coding RNAs and their influence on splicing phenomenon, and (2) the post-transcription level by modulating the regulatory functions of ribonucleoproteins and RNA binding proteins in mRNA translation, dendritic translocation as well as protein synthesis and synaptic turnover. DNA methylation changes are well recognized and highly correlated to gene expression levels as well as, learning and memory; however, RNA methylation changes are recently characterized and yet their functional implications are not established. This review article provides some insight on the intriguing consequences of changes in methylation levels on mRNA life-cycle. We also suggest that, since methylation is under the control of glutathione anti-oxidant levels (Lertratanangkoon et al., 1997), the redox status of

  7. Nerve Growth Factor Increases mRNA Levels for the Prion Protein and the β -amyloid Protein Precursor in Developing Hamster Brain

    Science.gov (United States)

    Mobley, William C.; Neve, Rachael L.; Prusiner, Stanley B.; McKinley, Michael P.

    1988-12-01

    Deposition of amyloid filaments serves as a pathologic hallmark for some neurodegenerative disorders. The prion protein (PrP) is found in amyloid of animals with scrapie and humans with Creutzfeldt-Jakob disease; the β protein is present in amyloid deposits in Alzheimer disease and Down syndrome patients. These two proteins are derived from precursors that in the brain are expressed primarily in neurons and are membrane bound. We found that gene expression for PrP and the β -protein precursor (β -PP) is regulated in developing hamster brain. Specific brain regions showed distinct patterns of ontogenesis for PrP and β -PP mRNAs. The increases in PrP and β -PP mRNAs in developing basal forebrain coincided with an increase in choline acetyltransferase activity, raising the possibility that these markers might be coordinately controlled in cholinergic neurons and regulated by nerve growth factor (NGF). Injections of NGF into the brains of neonatal hamsters increased both PrP and β -PP mRNA levels. Increased PrP and β -PP mRNA levels induced by NGF were confined to regions that contain NGF-responsive cholinergic neurons and were accompanied by elevations in choline acetyltransferase. It remains to be established whether or not exogenous NGF acts to increase PrP and β -PP gene expression selectively in forebrain cholinergic neurons in the developing hamster and endogenous NGF regulates expression of these genes.

  8. Analysis of expression of secreted phospholipases A2 in mouse tissues at protein and mRNA levels.

    Science.gov (United States)

    Eerola, Leena I; Surrel, Fanny; Nevalainen, Timo J; Gelb, Michael H; Lambeau, Gérard; Laine, V Jukka O

    2006-07-01

    Secreted phospholipases A(2) (sPLA(2)) form a group of low-molecular weight enzymes that catalyze the hydrolysis of phospholipids. Some sPLA(2)s are likely to play a role in inflammation, cancer, and as antibacterial enzymes in innate immunity. We developed specific and sensitive time-resolved fluroimmunoassays (TR-FIA) for mouse group (G) IB, GIIA, GIID, GIIE, GIIF, GV and GX sPLA(2)s and measured their concentrations in mouse serum and tissues obtained from both Balb/c and C57BL/6J mice. We also analyzed the mRNA expression of the sPLA(2)s by quantitative real-time reverse transcriptase PCR (qPCR). In most tissues, the concentrations of sPLA(2) proteins corresponded to the expression of sPLA(2)s at the mRNA level. With a few exceptions, the sPLA(2) proteins were found in the gastrointestinal tract. The qPCR results showed that GIB sPLA(2) is synthesized widely in the gastrointestinal tract, including esophagus and colon, in addition to stomach and pancreas. Our results also suggest that the loss of GIIA sPLA(2) in the intestine of GIIA sPLA(2)-deficient C57BL/6J mice is not compensated by other sPLA(2)s under normal conditions. Outside the gastrointestinal tract, sPLA(2)s were expressed occasionally in a number of tissues. The TR-FIAs developed in the current study may serve as useful tools to measure the levels of sPLA(2) proteins in mouse serum and tissues in various experimental settings.

  9. Regulation of CD163 mRNA and soluble CD163 protein in human adipose tissue in vitro.

    Science.gov (United States)

    Fjeldborg, Karen; Møller, Holger J; Richelsen, Bjørn; Pedersen, Steen B

    2014-10-01

    CD163-positive macrophages are highly expressed in the human adipose tissue (AT) particularly from obese individuals. However, little is known about the regulation of CD163 mRNA and the protein level of sCD163 in human AT. We aimed to examine the regulation of CD163 and sCD163 in AT. Human s.c. AT samples (n=5) were stimulated with dexamethasone (DEX; 200  nmol/l), lipopolysaccharide (LPS; 100  ng/ml), or DEX+LPS for various time periods up to 24  h. Gene expressions of CD163, ADAM17, IL10, and TNFA (TNF) were measured by RT-PCR. Protein levels of sCD163, IL10, and TNFα (TNF) were measured by ELISA. Furthermore, AT was separated into stromal and adipocyte fraction. We found that CD163 mRNA was strongly expressed in the stromal vascular fraction but hardly detectable in the isolated adipocytes. Incubating whole AT with DEX significantly up-regulated CD163 (PCD163 (P>0.05). By contrast, the protein level of sCD163 was not affected by DEX (P>0.05), but LPS significantly increased the level of sCD163 and TNFα (PCD163 and the release of sCD163 are differentially regulated in human AT. Moreover, similar to studies on differentiated blood monocytes, TNFα and sCD163 are concomitantly released in human AT by LPS, which also up-regulate ADAM17. © 2014 Society for Endocrinology.

  10. Mapping of the Mouse Actin Capping Protein Beta Subunit Gene

    Directory of Open Access Journals (Sweden)

    Cooper John A

    2000-07-01

    Full Text Available Abstract Background Capping protein (CP, a heterodimer of α and β subunits, is found in all eukaryotes. CP binds to the barbed ends of actin filaments in vitro and controls actin assembly and cell motility in vivo. Vertebrates have three isoforms of CPβ produced by alternatively splicing from one gene; lower organisms have one gene and one isoform. Results We isolated genomic clones corresponding to the β subunit of mouse CP and identified its chromosomal location by interspecies backcross mapping. Conclusions The CPβ gene (Cappb1 mapped to Chromosome 4 between Cdc42 and D4Mit312. Three mouse mutations, snubnose, curly tail, and cribriform degeneration, map in the vicinity of the β gene.

  11. Transforming growth factor beta stimulation of biglycan gene expression is potentially mediated by sp1 binding factors

    DEFF Research Database (Denmark)

    Heegaard, Anne-Marie; Xie, Zhongjian; Young, Marian Frances;

    2004-01-01

    Biglycan is a small leucine-rich proteoglycan which is localized in the extracellular matrix of bone and other specialized connective tissues. Both biglycan mRNA and protein are up-regulated by transforming growth factor-beta(1) (TGF-beta(1)) and biglycan appears to influence TGF-beta(1) activity...

  12. Determinants of the rate of mRNA translocation in bacterial protein synthesis.

    Science.gov (United States)

    Borg, Anneli; Ehrenberg, Måns

    2015-05-08

    Studying the kinetics of translocation of mRNA and tRNAs on the translating ribosome is technically difficult since the rate-limiting steps involve large conformational changes without covalent bond formation or disruption. Here, we have developed a unique assay system for precise estimation of the full translocation cycle time at any position in any type of open reading frame (ORF). Using a buffer system optimized for high accuracy of tRNA selection together with high concentration of elongation factor G, we obtained in vivo compatible translocation rates. We found that translocation was comparatively slow early in the ORF and faster further downstream of the initiation codon. The maximal translocation rate decreased from the in vivo compatible value of 30 s(-1) at 1 mM free Mg2+ concentration to the detrimentally low value of 1 s(-1) at 6 mM free Mg2+ concentration. Thus, high and in vivo compatible accuracy of codon translation, as well as high and in vivo compatible translocation rate, required a remarkably low Mg2+ concentration. Finally, we found that the rate of translocation deep inside an ORF was not significantly affected upon variation of the standard free energy of interaction between a 6-nt upstream Shine-Dalgarno (SD)-like sequence and the anti-SD sequence of 16S rRNA in a range of 0-6 kcal/mol. Based on these experiments, we discuss the optimal choice of Mg2+ concentration for maximal fitness of the living cell by taking its effects on the accuracy of translation, the peptide bond formation rate and the translocation rate into account.

  13. Virus-Like Particles of mRNA with Artificial Minimal Coat Proteins

    NARCIS (Netherlands)

    Jekhmane, Shehrazade; Haas, De Rob; Paulino Da Silva Filho, Omar; Asbeck, van Alexander H.; Favretto, Marco Emanuele; Hernandez Garcia, Armando; Brock, Roland; Vries, De Renko

    2017-01-01

    RNA has enormous potential as a therapeutic, yet, the successful application depends on efficient delivery strategies. In this study, we demonstrate that a designed artificial viral coat protein, which self-assembles with DNA to form rod-shaped virus-like particles (VLPs), also encapsulates and

  14. Isolation, purification and characterization of beta-1,3-glucan binding protein from the plasma of marine mussel Perna viridis.

    Science.gov (United States)

    Jayaraj, S S; Thiagarajan, R; Arumugam, M; Mullainadhan, P

    2008-06-01

    A beta-1,3-glucan binding protein (betaGBP) specific for laminarin (a beta-1,3-glucan) was detected for the first time in a mollusc, Perna viridis. betaGBP was isolated and purified from the plasma using laminarin precipitation and affinity chromatography on laminarin-Sepharose 6B, respectively. It agglutinated bakers yeast, bacteria, and erythrocytes and enhanced prophenoloxidase (proPO) activity of the plasma in a dose-dependent manner. The purified betaGBP appeared as a single band in native-PAGE and the purity was conformed by HPLC. The protein has a molecular weight estimate of 510kDa as determined by SDS-PAGE and in isoelectric focusing the purified betaGBP was focused as a single band at pI 5.3. betaGBP was found to possess inherent serine protease activity but lacked beta-1,3-glucanase activity and all these results suggest that plasma betaGBP of P. viridis functions as a recognition molecule for beta-1,3-glucan on the surface of microbial cell walls. This recognition and binding lead to the activation of the prophenoloxidase cascade mediated by the inherent serine protease activity of betaGBP. Presence of agglutinating activity and serine protease activity shows that betaGBP is a bifunctional protein. The findings are discussed in light of the importance of this protein in the innate immune response of P. viridis, and they implicate evolutionary link with similar proteins found in other invertebrates.

  15. AMP-activated protein kinase (AMPK mediates nutrient regulation of thioredoxin-interacting protein (TXNIP in pancreatic beta-cells.

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    Maayan Shaked

    Full Text Available Thioredoxin-interacting protein (TXNIP regulates critical biological processes including inflammation, stress and apoptosis. TXNIP is upregulated by glucose and is a critical mediator of hyperglycemia-induced beta-cell apoptosis in diabetes. In contrast, the saturated long-chain fatty acid palmitate, although toxic to the beta-cell, inhibits TXNIP expression. The mechanisms involved in the opposing effects of glucose and fatty acids on TXNIP expression are unknown. We found that both palmitate and oleate inhibited TXNIP in a rat beta-cell line and islets. Palmitate inhibition of TXNIP was independent of fatty acid beta-oxidation or esterification. AMP-activated protein kinase (AMPK has an important role in cellular energy sensing and control of metabolic homeostasis; therefore we investigated its involvement in nutrient regulation of TXNIP. As expected, glucose inhibited whereas palmitate stimulated AMPK. Pharmacologic activators of AMPK mimicked fatty acids by inhibiting TXNIP. AMPK knockdown increased TXNIP expression in presence of high glucose with and without palmitate, indicating that nutrient (glucose and fatty acids effects on TXNIP are mediated in part via modulation of AMPK activity. TXNIP is transcriptionally regulated by carbohydrate response element-binding protein (ChREBP. Palmitate inhibited glucose-stimulated ChREBP nuclear entry and recruitment to the Txnip promoter, thereby inhibiting Txnip transcription. We conclude that AMPK is an important regulator of Txnip transcription via modulation of ChREBP activity. The divergent effects of glucose and fatty acids on TXNIP expression result in part from their opposing effects on AMPK activity. In light of the important role of TXNIP in beta-cell apoptosis, its inhibition by fatty acids can be regarded as an adaptive/protective response to glucolipotoxicity. The finding that AMPK mediates nutrient regulation of TXNIP may have important implications for the pathophysiology and treatment

  16. Regional induction of c-fos and heat shock protein-72 mRNA following fluid-percussion brain injury in the rat

    Energy Technology Data Exchange (ETDEWEB)

    Raghupathi, R.; Welsh, F.A.; Gennarelli, T.A. [Univ. of Pennsylvania, Philadelphia, PA (United States)] [and others

    1995-05-01

    To evaluate the cellular response to traumatic brain injury, the expression of mRNA for c-fos and the 72-kDa heat shock protein (hsp72) was determined using in situ hybridization following lateral fluid-percussion injury (2.2-2.4 atm) in rat brain. At 2 h after injury, induction of c-fos mRNA was restricted to regions of the cortex surrounding the contusion area. An increase in c-fos mRNA, but not hsp72 mRNA, was observed bilaterally in the CA{sub 3} subfield of the hippocampus and the granule cells of the dentate gyrus and in the thalamus ipsilateral to the impact site. By 6 h, increased expression of c-fos mRNA was observed only in the corpus callosum on the impact side; hsp72 mRNA persisted in the deep cortical layers and upper layers of the subcortical white matter below the site of maximal injury. By 24 h, both c-fos and hsp72 mRNA had returned to control levels in all regions of the brain. These results demonstrate that lateral fluid-percussion brain injury triggers regionally and temporally specific expression of c-fos and hsp72 mRNA, which may be suggestive of differential neurochemical alterations in neurons and glia following experimental brain injury. 33 refs., 3 figs., 1 tab.

  17. Real-time RT-PCR analysis of mRNA decay: half-life of Beta-actin mRNA in human leukemia CCRF-CEM and Nalm-6 cell lines

    Directory of Open Access Journals (Sweden)

    Barredo Julio C

    2002-03-01

    Full Text Available Abstract Background We describe an alternative method to determine mRNA half-life (t1/2 based on the Real-Time RT-PCR procedure. This approach was evaluated by using the β-actin gene as a reference molecule for measuring of mRNA stability. Results Human leukemia Nalm-6 and CCRF-CEM cells were treated with various concentrations of Actinomycin D to block transcription and aliquots were removed periodically. Total RNA was isolated and quantified using the RiboGreen® fluorescent dye with the VersaFluor Fluorometer System. One μg of total RNA was reverse transcribed and used as template for the amplification of a region of the β-actin gene (231 bp. To generate the standard curve, serial ten-fold dilutions of the pBactin-231 vector containing the cDNA amplified fragment were employed, β-actin mRNAs were quantified by Real-Time RT-PCR using the SYBR® Green I fluorogenic dye and data analyzed using the iCycle iQ system software. Using this method, the β-actin mRNA exhibited a half-life of 6.6 h and 13.5 h in Nalm-6 and CCRF-CEM cells, respectively. The t1/2 value obtained for Nalm-6 is comparable to those estimated from Northern blot studies, using normal human leukocytes (5.5 h. Conclusions We have developed a rapid, sensitive, and reliable method based on Real-Time RT-PCR for measuring mRNA half-life. Our results confirm that β-actin mRNA half-life can be affected by the cellular growth rate.

  18. The CCAAT/enhancer-binding protein beta-2 isoform (CEBPβ-2 upregulates galectin-7 expression in human breast cancer cells.

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    Carole G Campion

    Full Text Available Galectin-7 is considered a gene under the control of p53. However, elevated expression of galectin-7 has been reported in several forms of cancer harboring an inactive p53 pathway. This is especially true for breast cancer where galectin-7 expression is readily expressed in a high proportion in basal-like breast cancer tissues, conferring cancer cells with increased resistance to cell death and metastatic properties. These observations suggest that other transcription factors are capable of inducing galectin-7 expression. In the present work, we have examined the role of CCAAT/enhancer-binding protein beta (C/EBPβ in inducing expression of galectin-7. C/EBP proteins have been shown to contribute to breast cancer by upregulating pro-metastatic genes. We paid particular attention to C/EBPβ-2 (also known as LAP2, the most transcriptionally active of the C/EBPβ isoforms. Our results showed that ectopic expression of C/EBPβ-2 in human breast cancer cells was sufficient to induce expression of galectin-7 at both the mRNA and protein levels. In silico analysis further revealed the presence of an established CEBP element in the galectin-7 promoter. Mutation of this binding site abolished the transcriptional activity of the galectin-7 promoter. Chromatin immunoprecipitation analysis confirmed that C/EBPβ-2 binds to the endogenous galectin-7 promoter. Analysis of galectin-7 protein expression in normal epithelia and in breast carcinoma by immunohistochemistry further showed the expression pattern of C/EBPβ closely micmicked that of galectin-7, most notably in mammary myoepithelial cells and basal-like breast cancer where galectin-7 is preferentially expressed. Taken together, our findings suggest that C/EBPβ is an important mediator of galectin-7 gene activation in breast cancer cells and highlight the different transcriptional mechanisms controlling galectin-7 in cancer cells.

  19. EFFECTS OF QUERCETIN ON CELL MORPHOLOGY AND EXPRESSION OF PML mRNA AND PROTEIN OF NB4 AND HL-60 CELLS

    Institute of Scientific and Technical Information of China (English)

    钟璐; 陈芳源; 韩洁英; 邵念贤; 欧阳仁荣

    2001-01-01

    Objective To investigate the effects of quercetin on cell morphology, expression of promyelocytic leukemia ( PML ) mRNA and PML protein localization of NB4, HL-60 cells. Methods Cells morphology assayed by Wright's stain, fluorescence stain, and PML mRNA expression by RT-PCR , PML protein localization by immuno-fluorescence. Results The typical apoptosis was found in NB4 and HL-60 cells after treatment with quercetin . Immuno-fluorescence analysis showed after treatment with quercetin , the fusion protein disappeared in NB4 cells, PML protein relocated, then degraded, and that also seen in HL-60 cells. The expression of PML mRNA is not changed in quercetin-treated cells. Conclusion PML play the role of apoptosis inducer in leukemia cells at the translational level, quercetin can inhibit the proliferation of leukemia cells, and induce NB4, HL-60

  20. Influence of genetic polymorphisms of multidrug and toxin extrusion protein 1 on its mRNA expression in peripheral blood cells

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    Hitoshi Ando

    2016-06-01

    Full Text Available This study aimed to determine the effect of multidrug and toxin extrusion protein 1 (MATE1 genetic variants on its transcript expression in peripheral blood cells. Consistent with previous in vitro findings, MATE1 mRNA levels were significantly higher in subjects carrying rs2453579, but not rs2252281, compared to those without either of these promoter variants. In addition, the mRNA levels did not differ between subjects with both variants and those with neither allele. Thus, this study reveals that the influence of MATE1 genetic variants on its mRNA expression can be detected in vivo using peripheral blood.

  1. Role of a redox-based methylation switch in mRNA life cycle ( pre- & post- transcriptional maturation and protein turnover : Implications in neurological disorders

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    MALAV SUCHIN TRIVEDI

    2012-06-01

    Full Text Available Homeostatic synaptic scaling in response to neuronal stimulus or activation, as well as due to changes in cellular niche, is an important phenomenon for memory consolidation, retrieval, and other similar cognitive functions. Neurological disorders and cognitive disabilities in autism, Rett syndrome, schizophrenia, dementia etc., are strongly correlated to alterations in protein expression (both synaptic and cytoplasmic. This correlation suggests that efficient temporal regulation of synaptic protein expression is important for synaptic plasticity. In addition, equilibrium between mRNA processing, protein translation and protein turnover is a critical sensor/trigger for recording synaptic information, normal cognition and behavior. Thus a regulatory switch, controlling the lifespan, maturation and processing of mRNA, might influence cognition and adaptive behavior. Here, we propose a two part novel hypothesis that methylation might act as this suggested coordinating switch to critically regulate mRNA maturation at 1.The pre-transcription level, by regulating precursor-RNA (pre-RNA processing into mRNA, via other non-coding RNAs and their influence on splicing phenomenon, and 2. the post-transcription level by modulating the regulatory functions of ribonucleoproteins (RNP and RNA binding proteins (RNABP in mRNA translation, dendritic translocation as well as protein synthesis and synaptic turnover. DNA methylation changes are well recognized and highly correlated to gene expression levels as well as, learning and memory; however, RNA methylation changes are recently characterized and yet their functional implications are not established. This review article provides some insight on the intriguing consequences of changes in methylation levels on mRNA life-cycle. We also suggest that, since methylation is under the control of glutathione antioxidant levels, the redox status of neurons might be the central regulatory switch for methylation

  2. Bone morphogenetic protein-2 antagonizes renal interstitial fibrosis by promoting catabolism of type I transforming growth factor-beta receptors.

    Science.gov (United States)

    Yang, Yu-Lin; Liu, Yi-Shiuan; Chuang, Lea-Yea; Guh, Jinn-Yuh; Lee, Tao-Chen; Liao, Tung-Nan; Hung, Min-Yuan; Chiang, Tai-An

    2009-02-01

    TGF-beta is a therapeutic target for renal fibrosis. Scientists have long sought ways to antagonize TGF-beta to ameliorate diabetic nephropathy. Bone morphogenetic protein (BMP-2) is a member of the TGF-beta superfamily and is highly regulated in the kidney. Thus, the role of BMP-2 was investigated in NRK-49F cells (rat fibroblasts). We showed that TGF-beta1 induces an increase in fibronectin. Treatment with exogenous BMP-2 or pCMV-BMP-2 significantly reversed the TGF-beta1-induced increase in fibronectin concomitant with a significant decrease in type I TGF-beta receptors (TGF-beta RI). Moreover, BMP-2 significantly shortened the half-life of TGF-beta RI. These results are related to proteosomal activation because MG132, a proteasome inhibitor, abolished BMP-2-mediated degradation of TGF-beta RI. This was confirmed because BMP-2 time course dependently enhanced the ubiquitination level of TGF-beta RI. In addition, Smads would seem to be involved in the interaction of BMP-2 and TGF-beta. We demonstrated that BMP-2 significantly reversed the TGF-beta1-induced increase in pSmad2/3 and reversed the TGF-beta1-induced decrease in inhibitory Smad7. Most importantly, Smad7 small interfering RNA abolished the BMP-2-induced decrease in TGF-beta RI. We evaluated the clinical efficacy of BMP-2 using unilateral ureteral obstruction rats. BMP-2 was administered ip for 7 d. In the unilateral ureteral obstruction kidneys, interstitial fibrosis was prominent. However, treatment with BMP-2 dramatically reduced Masson's trichrome staining (collagen) in the interstitial and tubular areas of the kidneys concomitantly with a reduction in TGF-beta RI. These results suggest that BMP-2 acts as a novel fibrosis antagonizing cytokine partly by down-regulating TGF-beta RI and Smads.

  3. Identification of an RNA-binding protein that is phosphorylated by PTH and potentially mediates PTH-induced destabilization of Npt2a mRNA.

    Science.gov (United States)

    Murray, Rebecca D; Merchant, Michael L; Hardin, Ericka; Clark, Barbara; Khundmiri, Syed J; Lederer, Eleanor D

    2016-02-01

    Parathyroid hormone (PTH) is a key regulator of the expression and function of the type IIa sodium-phosphate cotransporter (Npt2a), the protein responsible for regulated renal phosphate reabsorption. We previously showed that PTH induces rapid decay of Npt2a mRNA through posttranscriptional mechanisms. We hypothesized that PTH-induced changes in RNA-binding protein (RBP) activity mediate the degradation of Npt2a mRNA. To address this aim, we treated opossum kidney (OK) cells, a PTH-sensitive proximal tubule cell culture model, with 100 nM PTH for 30 min and 2 h, followed by mass spectrometry characterization of the PTH-stimulated phosphoproteome. We identified 1,182 proteins differentially phosphorylated in response to PTH, including 68 RBPs. Preliminary analysis identified a phospho-RBP, hnRNPK-homology-type-splicing regulatory protein (KSRP), with predicted binding sites for the 3'-untranslated region (UTR) of Npt2a mRNA. Western blot analysis confirmed expression of KSRP in OK cells and showed PTH-dependent translocation to the nucleus. Immunoprecipitation of KSRP from control and PTH-treated cells followed by RNA isolation and RT-quantitative PCR analysis identified Npt2a mRNA from both control and PTH-treated KSRP pulldowns. Knockdown of KSRP followed by PTH treatment showed that KSRP is required for mediating PTH-stimulated reduction in sodium/hydrogen exchanger 3 mRNA, but not Npt2a mRNA. We conclude that 1) PTH is a major regulator of both transcription and translation, and 2) KSRP binds Npt2a mRNA but its role in PTH regulation of Npt2a mRNA is not clear.

  4. Muscle-specific differences in the response of mitochondrial proteins to beta-GPA feeding: an evaluation of potential mechanisms.

    Science.gov (United States)

    Williams, Deon B; Sutherland, Lindsey N; Bomhof, Marc R; Basaraba, Susan A U; Thrush, A Brianne; Dyck, David J; Field, Catherine J; Wright, David C

    2009-06-01

    Beta-Guanadinopropionic acid (beta-GPA) feeding leads to reductions in skeletal muscle phosphagen concentrations and has been used as a tool with which to study the effects of energy charge on skeletal muscle metabolism. Supplementing standard rodent diets with beta-GPA leads to increases in mitochondrial enzyme content in fast but not slow-twitch muscles from male rats. Given this apparent discrepancy between muscle types we used beta-GPA feeding as a model to study signaling pathways involved in mitochondrial biogenesis. We hypothesized that beta-GPA feeding would result in a preferential activation of p38 MAPK and AMPK signaling and reductions in RIP140 protein content in triceps but not soleus muscle. Despite similar reductions in high-energy phosphate concentrations, 6 wk of beta-GPA feeding led to increases in mitochondrial proteins in triceps but not soleus muscles. Differences in the response of mitochondrial proteins to beta-GPA feeding did not seem to be related to a differential activation of p38 MAPK and AMPK signaling pathways or discrepancies in the induction of PPARgamma coactivator (PGC)-1alpha and -1beta. The protein content and expression of the nuclear corepressor RIP140 was reduced in triceps but not soleus muscle. Collectively our results indicate that chronic reductions in high-energy phosphates lead to the activation of p38 MAPK and AMPK signaling and increases in the expression of PGC-1alpha and -1beta in both soleus and triceps muscles. The lack of an effect of beta-GPA feeding on mitochondrial proteins in the soleus muscles could be related to a fiber type-specific effect of beta-GPA on RIP140 protein content.

  5. Modulation of the beta-catenin signaling pathway by the dishevelled-associated protein Hipk1.

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    Sarah H Louie

    Full Text Available BACKGROUND: Wnts are evolutionarily conserved ligands that signal through beta-catenin-dependent and beta-catenin-independent pathways to regulate cell fate, proliferation, polarity, and movements during vertebrate development. Dishevelled (Dsh/Dvl is a multi-domain scaffold protein required for virtually all known Wnt signaling activities, raising interest in the identification and functions of Dsh-associated proteins. METHODOLOGY: We conducted a yeast-2-hybrid screen using an N-terminal fragment of Dsh, resulting in isolation of the Xenopus laevis ortholog of Hipk1. Interaction between the Dsh and Hipk1 proteins was confirmed by co-immunoprecipitation assays and mass spectrometry, and further experiments suggest that Hipk1 also complexes with the transcription factor Tcf3. Supporting a nuclear function during X. laevis development, Myc-tagged Hipk1 localizes primarily to the nucleus in animal cap explants, and the endogenous transcript is strongly expressed during gastrula and neurula stages. Experimental manipulations of Hipk1 levels indicate that Hipk1 can repress Wnt/beta-catenin target gene activation, as demonstrated by beta-catenin reporter assays in human embryonic kidney cells and by indicators of dorsal specification in X. laevis embryos at the late blastula stage. In addition, a subset of Wnt-responsive genes subsequently requires Hipk1 for activation in the involuting mesoderm during gastrulation. Moreover, either over-expression or knock-down of Hipk1 leads to perturbed convergent extension cell movements involved in both gastrulation and neural tube closure. CONCLUSIONS: These results suggest that Hipk1 contributes in a complex fashion to Dsh-dependent signaling activities during early vertebrate development. This includes regulating the transcription of Wnt/beta-catenin target genes in the nucleus, possibly in both repressive and activating ways under changing developmental contexts. This regulation is required to modulate gene

  6. The identification of allergen proteins in sugar beet (Beta vulgaris pollen causing occupational allergy in greenhouses

    Directory of Open Access Journals (Sweden)

    Blomqvist Anna

    2008-08-01

    Full Text Available Abstract Background During production of sugar beet (Beta vulgaris seeds in greenhouses, workers frequently develop allergic symptoms. The aim of this study was to identify and characterize possible allergens in sugar beet pollen. Methods Sera from individuals at a local sugar beet seed producing company, having positive SPT and specific IgE to sugar beet pollen extract, were used for immunoblotting. Proteins in sugar beet pollen extracts were separated by 1- and 2-dimensional electrophoresis, and IgE-reactive proteins analyzed by liquid chromatography tandem mass spectrometry. Results A 14 kDa protein was identified as an allergen, since IgE-binding was inhibited by the well-characterized allergen Che a 2, profilin, from the related species Chenopodium album. The presence of 17 kDa and 14 kDa protein homologues to both the allergens Che a 1 and Che a 2 were detected in an extract from sugar beet pollen, and partial amino acid sequences were determined, using inclusion lists for tandem mass spectrometry based on homologous sequences. Conclusion Two occupational allergens were identified in sugar beet pollen showing sequence similarity with Chenopodium allergens. Sequence data were obtained by mass spectrometry (70 and 25%, respectively for Beta v 1 and Beta v 2, and can be used for cloning and recombinant expression of the allergens. As for treatment of Chenopodium pollinosis, immunotherapy with sugar beet pollen extracts may be feasible.

  7. Discrepancy between mRNA and protein abundance: Insight from information retrieval process in computers

    Science.gov (United States)

    Wang, Degeng

    2008-01-01

    Discrepancy between the abundance of cognate protein and RNA molecules is frequently observed. A theoretical understanding of this discrepancy remains elusive, and it is frequently described as surprises and/or technical difficulties in the literature. Protein and RNA represent different steps of the multi-stepped cellular genetic information flow process, in which they are dynamically produced and degraded. This paper explores a comparison with a similar process in computers - multi-step information flow from storage level to the execution level. Functional similarities can be found in almost every facet of the retrieval process. Firstly, common architecture is shared, as the ribonome (RNA space) and the proteome (protein space) are functionally similar to the computer primary memory and the computer cache memory respectively. Secondly, the retrieval process functions, in both systems, to support the operation of dynamic networks – biochemical regulatory networks in cells and, in computers, the virtual networks (of CPU instructions) that the CPU travels through while executing computer programs. Moreover, many regulatory techniques are implemented in computers at each step of the information retrieval process, with a goal of optimizing system performance. Cellular counterparts can be easily identified for these regulatory techniques. In other words, this comparative study attempted to utilize theoretical insight from computer system design principles as catalysis to sketch an integrative view of the gene expression process, that is, how it functions to ensure efficient operation of the overall cellular regulatory network. In context of this bird’s-eye view, discrepancy between protein and RNA abundance became a logical observation one would expect. It was suggested that this discrepancy, when interpreted in the context of system operation, serves as a potential source of information to decipher regulatory logics underneath biochemical network operation. PMID

  8. Effects of Raloxifene on Caveolin-1 mRNA and Protein Expressions in Vascular Smooth Muscle Cells

    Institute of Scientific and Technical Information of China (English)

    Fa-Lin YANG; Hong HE; Xian-Xi LIU; Bing TU; Xian-Wei ZENG; Ji-Xin SU; Xin WANG; Qin HU

    2006-01-01

    Caveolin-1 is regulated by estrogen in vascular smooth muscle cells. Raloxifene, a selectiveestrogen receptor modulator that possibly has cardioprotective properties without an increased risk of c ancer or other side effects of estrogen, may be used in women with risk of coronary artery disease. However, the relationship between raloxifene and caveolin-1 is still unknown. Therefore, this study was designed to see whether raloxifene regulates caveolin- 1 expression and if so, whether such regulation is mediated by estrogen receptor. Rat aortic smooth muscle cells were cultured in the absence or presence of raloxifene (10-8 to 10-6 M) for 12 or 24 h. Both mRNA and protein levels of caveolin-1 were increased significantly after 24 h treatment with raloxifene. These increases were inhibited by estrogen receptor antagonist ICI 182780 (10-5 M). Results of this study suggest that raloxifene stimulates caveolin- 1 transcription and translation through estrogen receptor mediated mechanisms.

  9. EGF receptor inhibitors increase ErbB3 mRNA and protein levels in breast cancer cells

    DEFF Research Database (Denmark)

    Grøvdal, Lene Melsæther; Kim, Jiyoung; Holst, Mikkel Roland

    2012-01-01

    to EGFR inhibitor treatment in ErbB2 overexpressing breast cancer cells. We found that gefitinib treatment increased ErbB3 expression, both at protein and mRNA levels. ErbB3 expression was upregulated not only by gefitinib but also by a panel of different EGFR inhibitors, suggesting that inhibition......The potential benefits of drugs directly targeting the ErbB receptors for cancer therapy have led to an extensive development within this field. However, the clinical effects of ErbB receptor-targeting drugs in cancer treatment are limited due to a high frequency of resistance. It has been reported...... that, when inhibiting the epidermal growth factor receptor (EGFR) with the tyrosine kinase inhibitor gefitinib, increased activation of ErbB3 via MET, or by re-localization of ErbB3 mediates cell survival. Here we show further evidence that members of the ErbB receptor family facilitate resistance...

  10. Lyophilization-induced protein denaturation in phosphate buffer systems: monomeric and tetrameric beta-galactosidase.

    Science.gov (United States)

    Pikal-Cleland, K A; Carpenter, J F

    2001-09-01

    During freezing in phosphate buffers, selective precipitation of a less soluble buffer component and subsequent pH shifts may induce protein denaturation. Previous reports indicate significantly more inactivation and secondary structural perturbation of monomeric and tetrameric beta-galactosidase (beta-gal) during freeze-thawing in sodium phosphate (NaP) buffer as compared with potassium phosphate (KP) buffer. This observation was attributed to the significant pH shifts (from 7.0 to as low as 3.8) observed during freezing in the NaP buffer (1). In the current study, we investigated the impact of the additional stress of dehydration after freezing on the recovery of active protein on reconstitution and the retention of the native structure in the dried state. Freeze-drying monomeric and tetrameric beta-gal in either NaP or KP buffer resulted in significant secondary structural perturbations, which were greatest for the NaP samples. However, similar recoveries of active monomeric protein were observed after freeze-thawing and freeze-drying, indicating that most dehydration-induced unfolding was reversible on reconstitution of the freeze-dried protein. In contrast, the tetrameric protein was more susceptible to dehydration-induced denaturation as seen by the greater loss in activity after reconstitution of the freeze-dried samples relative to that measured after freeze-thawing. To ensure optimal protein stability during freeze-drying, the protein must be protected from both freezing and dehydration stresses. Although poly(ethylene glycol) and dextran are preferentially excluded solutes and should confer protection during freezing, they were unable to prevent lyophilization-induced denaturation. In addition, Tween did not foster maintenance of native protein during freeze-drying. However, sucrose, which hydrogen bonds to dried protein in the place of lost water, greatly reduced freezing- and drying-induced denaturation, as observed by the high retention of native

  11. Effects of different doses of acitretin on FGF10 mRNA transcription and its protein translation in HaCaT cells

    Institute of Scientific and Technical Information of China (English)

    YU Chun-shui; TAN Sheng-shun; SUI Wei-chi; XI Yan-ping

    2006-01-01

    Objective:To observe the effects of different doses of acitretin on the transcription of FGF10 mRNA and the translation of FGF10 protein in cultured HaCaT cells. Methods: HaCaT cells were treated with different doses of acitretin for 48 h, then the changes on the transcription of FGF10 mRNA and the translation of FGF10 protein in these cells were detected by immunofluorescence and in situ hybridization assay. Results: Compared with the control group, the transcription of FGF10 mRNA and the translation of FGF10 protein were gradually decreased along with the increasing dose of acitretin. There were significant differences between different groups (P<0.01). Conclusion: Acitretin could inhibit the transcription of FGF10 mRNA and the translation of FGF10 protein in HaCaT cells. With the dose of acitretin increased, the stains of both FGF10 mRNA and FGF10 protein in HaCaT cells are reduced.

  12. Selection of random RNA fragments as method for searching for a site of regulation of translation of E. coli streptomycin mRNA by ribosomal protein S7.

    Science.gov (United States)

    Surdina, A V; Rassokhin, T I; Golovin, A V; Spiridonova, V A; Kraal, B; Kopylov, A M

    2008-06-01

    In E. coli cells ribosomal small subunit biogenesis is regulated by RNA-protein interactions involving protein S7. S7 initiates the subunit assembly interacting with 16S rRNA. During shift-down of rRNA synthesis level, free S7 inhibits self-translation by interacting with 96 nucleotides long specific region of streptomycin (str) mRNA between cistrons S12 and S7 (intercistron). Many bacteria do not have the extended intercistron challenging development of specific approaches for searching putative mRNA regulatory regions, which are able to interact with proteins. The paper describes application of SERF approach (Selection of Random RNA Fragments) to reveal regulatory regions of str mRNA. Set of random DNA fragments has been generated from str operon by random hydrolysis and then transcribed into RNA; the fragments being able to bind protein S7 (serfamers) have been selected by iterative rounds. S7 binds to single serfamer, 109 nucleotide long (RNA109), derived from the intercistron. After multiple copying and selection, the intercistronic mutant (RNA109) has been isolated; it has enhanced affinity to S7. RNA109 binds to the protein better than authentic intercistronic str mRNA; apparent dissociation constants are 26 +/- 5 and 60 +/- 8 nM, respectively. Location of S7 binding site on the mRNA, as well as putative mode of regulation of coupled translation of S12 and S7 cistrons have been hypothesized.

  13. Increased renal gene transcription of protein kinase C-beta in human diabetic nephropathy: relationship to long-term glycaemic control

    DEFF Research Database (Denmark)

    Langham, R.G.; Kelly, D.J.; Gow, R.M.;

    2008-01-01

    was examined in renal biopsies (n=25) with classical histological features of diabetic nephropathy and compared with that in normal control tissue (n=6). Peptide localisation of PKC-alpha, PKC-beta and the activated forms phosphorylated PKC-alpha and -beta was also performed on matched paraffin......-embedded sections of renal biopsies using immunohistochemistry. The effects of high glucose on PRKC-beta expression and peptide production in cultured human proximal tubular epithelial cells were assessed. RESULTS: Quantitative real-time PCR demonstrated a 9.9-fold increase in PRKC-beta mRNA in kidney biopsies...... quantitative analysis of mRNA from archival, formalin-fixed, paraffin-embedded tissue sections. RNA was extracted from scraped 6 microm sections of biopsy tissue, and PRKC-alpha and PRKC-beta (also known as PRKCA and PRKCB) mRNA measured using real-time PCR. Expression of genes encoding PKC isoforms...

  14. UPF1, a conserved nonsense-mediated mRNA decay factor, regulates cyst wall protein transcripts in Giardia lamblia.

    Directory of Open Access Journals (Sweden)

    Yi-Hsiu Chen

    Full Text Available The Giardia lamblia cyst wall is required for survival outside the host and infection. Three cyst wall protein (cwp genes identified to date are highly up-regulated during encystation. However, little is known of the molecular mechanisms governing their gene regulation. Messenger RNAs containing premature stop codons are rapidly degraded by a nonsense-mediated mRNA decay (NMD system to avoid production of non-functional proteins. In addition to RNA surveillance, NMD also regulates thousands of naturally occurring transcripts through a variety of mechanisms. It is interesting to know the NMD pathway in the primitive eukaryotes. Previously, we have found that the giardial homologue of a conserved NMD factor, UPF1, may be functionally conserved and involved in NMD and in preventing nonsense suppression. In this study, we tested the hypothesis that NMD factors can regulate some naturally occurring transcripts in G. lamblia. We found that overexpression of UPF1 resulted in a significant decrease of the levels of CWP1 and cyst formation and of the endogenous cwp1-3, and myb2 mRNA levels and stability. This indicates that NMD could contribute to the regulation of the cwp1-3 and myb2 transcripts, which are key to G. lamblia differentiation into cyst. Interestingly, we also found that UPF1 may be involved in regulation of eight other endogenous genes, including up-regulation of the translation elongation factor gene, whose product increases translation which is required for NMD. Our results indicate that NMD factor could contribute to the regulation of not only nonsense containing mRNAs, but also mRNAs of the key encystation-induced genes and other endogenous genes in the early-diverging eukaryote, G. lamblia.

  15. Beta-AMYLASE4, a noncatalytic protein required for starch breakdown, acts upstream of three active beta-amylases in Arabidopsis chloroplasts.

    Science.gov (United States)

    Fulton, Daniel C; Stettler, Michaela; Mettler, Tabea; Vaughan, Cara K; Li, Jing; Francisco, Perigio; Gil, Manuel; Reinhold, Heike; Eicke, Simona; Messerli, Gaëlle; Dorken, Gary; Halliday, Karen; Smith, Alison M; Smith, Steven M; Zeeman, Samuel C

    2008-04-01

    This work investigated the roles of beta-amylases in the breakdown of leaf starch. Of the nine beta-amylase (BAM)-like proteins encoded in the Arabidopsis thaliana genome, at least four (BAM1, -2, -3, and -4) are chloroplastic. When expressed as recombinant proteins in Escherichia coli, BAM1, BAM2, and BAM3 had measurable beta-amylase activity but BAM4 did not. BAM4 has multiple amino acid substitutions relative to characterized beta-amylases, including one of the two catalytic residues. Modeling predicts major differences between the glucan binding site of BAM4 and those of active beta-amylases. Thus, BAM4 probably lost its catalytic capacity during evolution. Total beta-amylase activity was reduced in leaves of bam1 and bam3 mutants but not in bam2 and bam4 mutants. The bam3 mutant had elevated starch levels and lower nighttime maltose levels than the wild type, whereas bam1 did not. However, the bam1 bam3 double mutant had a more severe phenotype than bam3, suggesting functional overlap between the two proteins. Surprisingly, bam4 mutants had elevated starch levels. Introduction of the bam4 mutation into the bam3 and bam1 bam3 backgrounds further elevated the starch levels in both cases. These data suggest that BAM4 facilitates or regulates starch breakdown and operates independently of BAM1 and BAM3. Together, our findings are consistent with the proposal that beta-amylase is a major enzyme of starch breakdown in leaves, but they reveal unexpected complexity in terms of the specialization of protein function.

  16. Small heat shock protein HspB8: its distribution in Alzheimer's disease brains and its inhibition of amyloid-beta protein aggregation and cerebrovascular amyloid-beta toxicity.

    NARCIS (Netherlands)

    Wilhelmus, M.M.; Boelens, W.C.; Otte-Holler, I.; Kamps, B.; Kusters, B.; Maat-Schieman, M.L.; Waal, R.M.W. de; Verbeek, M.M.

    2006-01-01

    Alzheimer's disease (AD) is characterized by pathological lesions, such as senile plaques (SPs) and cerebral amyloid angiopathy (CAA), both predominantly consisting of a proteolytic cleavage product of the amyloid-beta precursor protein (APP), the amyloid-beta peptide (Abeta). CAA is also the major

  17. Expression of NK1 receptor at the protein and mRNA level in the porcine female reproductive system.

    Science.gov (United States)

    Bukowski, R

    2014-01-01

    The presence and distribution of substance P (SP) receptor NK1 was studied in the ovary, the oviduct and the uterus (uterine horn and cervix) of the domestic pig using the methods of molecular biology (RT-PCR and immunoblot) and immunohistochemistry. The expression of NK1 receptor at mRNA level was confirmed with RT-PCR in all the studied parts of the porcine female reproductive system by the presence of 525 bp PCR product and at the protein level by the detection of 46 kDa protein band in immunoblot. Immunohistochemical staining revealed the cellular distribution of NK1 receptor protein. In the ovary NKI receptor was present in the wall of arterial blood vessels, as well as in ovarian follicles of different stages of development. In the tubular organs the NK1 receptor immunohistochemical stainings were observed in the wall of the arterial blood vessels, in the muscular membrane, as well as in the mucosal epithelium. The study confirmed the presence of NK1 receptor in the tissues of the porcine female reproductive tract which clearly points to the possibility that SP can influence porcine ovary, oviduct and uterus.

  18. Mapping the ribosomal protein S7 regulatory binding site on mRNA of the E. coli streptomycin operon.

    Science.gov (United States)

    Surdina, A V; Rassokhin, T I; Golovin, A V; Spiridonova, V A; Kopylov, A M

    2010-07-01

    In this work it is shown by deletion analysis that an intercistronic region (ICR) approximately 80 nucleotides in length is necessary for interaction with recombinant E. coli S7 protein (r6hEcoS7). A model is proposed for the interaction of S7 with two ICR sites-region of hairpin bifurcations and Shine-Dalgarno sequence of cistron S7. A de novo RNA binding site for heterologous S7 protein of Thermus thermophilus (r6hTthS7) was constructed by selection of a combinatorial RNA library based on E. coli ICR: it has only a single supposed protein recognition site in the region of bifurcation. The SERW technique was used for selection of two intercistronic RNA libraries in which five nucleotides of a double-stranded region, adjacent to the bifurcation, had the randomized sequence. One library contained an authentic AG (-82/-20) pair, while in the other this pair was replaced by AU. A serwamer capable of specific binding to r6hTthS7 was selected; it appeared to be the RNA68 mutant with eight nucleotide mutations. The serwamer binds to r6hTthS7 with the same affinity as homologous authentic ICR of str mRNA binds to r6hEcoS7; apparent dissociation constants are 89 +/- 43 and 50 +/- 24 nM, respectively.

  19. Integrative omics analysis. A study based on Plasmodium falciparum mRNA and protein data

    Science.gov (United States)

    2014-01-01

    Background Technological improvements have shifted the focus from data generation to data analysis. The availability of large amounts of data from transcriptomics, protemics and metabolomics experiments raise new questions concerning suitable integrative analysis methods. We compare three integrative analysis techniques (co-inertia analysis, generalized singular value decomposition and integrative biclustering) by applying them to gene and protein abundance data from the six life cycle stages of Plasmodium falciparum. Co-inertia analysis is an analysis method used to visualize and explore gene and protein data. The generalized singular value decomposition has shown its potential in the analysis of two transcriptome data sets. Integrative Biclustering applies biclustering to gene and protein data. Results Using CIA, we visualize the six life cycle stages of Plasmodium falciparum, as well as GO terms in a 2D plane and interpret the spatial configuration. With GSVD, we decompose the transcriptomic and proteomic data sets into matrices with biologically meaningful interpretations and explore the processes captured by the data sets. IBC identifies groups of genes, proteins, GO Terms and life cycle stages of Plasmodium falciparum. We show method-specific results as well as a network view of the life cycle stages based on the results common to all three methods. Additionally, by combining the results of the three methods, we create a three-fold validated network of life cycle stage specific GO terms: Sporozoites are associated with transcription and transport; merozoites with entry into host cell as well as biosynthetic and metabolic processes; rings with oxidation-reduction processes; trophozoites with glycolysis and energy production; schizonts with antigenic variation and immune response; gametocyctes with DNA packaging and mitochondrial transport. Furthermore, the network connectivity underlines the separation of the intraerythrocytic cycle from the gametocyte and

  20. Complexation of bovine beta-lactoglobulin with 11S protein fractions of soybean (Glycine max) and sesame (Sesamum indicum).

    Science.gov (United States)

    Anuradha, S N; Prakash, V

    2009-01-01

    Beta-lactoglobulin (beta-Lg) comprises 50% of the whey component of bovine milk. Protein-protein interactions between bovine beta-Lg and 11S protein fractions of soybean and sesame were investigated by turbidity, solubility behaviour and by evaluation of functional properties in the mixed systems. In this work, the aggregation behaviour of soybean and the whey protein (beta-Lg) showed the formation of soluble complexes. Turbidity and solubility studies showed that the proteins interacted at temperatures between 60 and 90+/-5 degrees C. Heating a mixture of beta-Lg and 11S proteins of soybean at higher temperatures formed soluble complexes with beta-Lg. It also reduced the self aggregation behaviour, especially that of 11S protein fraction of soybean. This reduced the precipitation of soybean proteins at higher temperature. The complex formed was resolved by gel filtration using high-performance liquid chromatography. Upon heating beta-Lg at neutral pH, native dimer starts to dissociate into monomers leading to the exposure of previously buried hydrophobic amino acids and the free thiol group. The soluble complex is formed by the exposed thiol groups. But interaction of beta-Lg with sesame 11S protein fractions did not form any soluble complexes. The mechanism of interaction indicates that hydrophobic interactions were preferred over disulfide linkages at the high salt concentrations of the buffer used. During thermal treatment the molecules are unfolded, leading to an exposure of the hydrophobic groups that further enhance the protein-protein interactions that are entropically driven hydrophobic interactions.

  1. Antimicrobial actions of the human epididymis 2 (HE2 protein isoforms, HE2alpha, HE2beta1 and HE2beta2

    Directory of Open Access Journals (Sweden)

    French Frank S

    2004-08-01

    Full Text Available Abstract Background The HE2 gene encodes a group of isoforms with similarities to the antimicrobial beta-defensins. We demonstrated earlier that the antimicrobial activity of HE2 proteins and peptides is salt resistant and structure dependent and involves permeabilization of bacterial membranes. In this study, we further characterize the antimicrobial properties of HE2 peptides in terms of the structural changes induced in E. coli and the inhibition of macromolecular synthesis. Methods E. coli treated with 50 micro g/ml of HE2alpha, HE2beta1 or HE2beta2 peptides for 30 and 60 min were visualized using transmission and scanning electron microscopy to investigate the impact of these peptides on bacterial internal and external structure. The effects of HE2alpha, HE2beta1 and HE2beta2 on E. coli macromolecular synthesis was assayed by incubating the bacteria with 2, 10 and 25 micro g/ml of the individual peptides for 0–60 min and measuring the incorporation of the radioactive precursors [methyl-3H]thymidine, [5-3H]uridine and L-[4,5-3H(N]leucine into DNA, RNA and protein. Statistical analyses using Student's t-test were performed using Sigma Plot software. Values shown are Mean ± S.D. Results E. coli treated with HE2alpha, HE2beta1 and HE2beta2 peptides as visualized by transmission electron microscopy showed extensive damage characterized by membrane blebbing, thickening of the membrane, highly granulated cytoplasm and appearance of vacuoles in contrast to the smooth and continuous membrane structure of the untreated bacteria. Similarly, bacteria observed by scanning electron microscopy after treating with HE2alpha, HE2beta1 or HE2beta2 peptides exhibited membrane blebbing and wrinkling, leakage of cellular contents, especially at the dividing septa, and external accumulation of fibrous materials. In addition, HE2alpha, HE2beta1 and HE2beta2 peptides inhibited E. coli DNA, RNA and protein synthesis. Conclusions The morphological changes observed

  2. A beta-D-galactoside binding protein from electric organ tissue of Electrophorus electricus.

    Science.gov (United States)

    Teichberg, V I; Silman, I; Beitsch, D D; Resheff, G

    1975-01-01

    Extracts of electric organ tissue of Electrophorus electricus contain a saccharide-binding protein, named electrolectin, which agglutinates trypsin-treated rabbit erythrocytes and is specifically inhibited by disaccharides containing nonreducing terminal beta-D-galactosyl residues. Electrolectin seems at least partially membrane-bound but is also found in soluble fractions of homoge-nates from which it can be purfied by affinity chromatography on cross-linked and desulfated agarose (ECD-Sepharose) as a protein of molecular weight 33,000. About 400 mg of electrolectin are present per kg of tissue. It has an affinity for lactose of 1.0 mM-1 and 5.5mM-1 as estimated, respectively, by hapten inhibition and fluorescence spectroscopy. Studies on the distribution of beta-D-galactoside-binding activity in animal tissues reveal particularly high levels in sheletal muscle tissue and in cultures of embryonic skeletal muscle and neuroblastoma cells. PMID:1055413

  3. Rapid, sequential activation of mitogen-activated protein kinases and transcription factors precedes proinflammatory cytokine mRNA expression in spleens of mice exposed to the trichothecene vomitoxin.

    Science.gov (United States)

    Zhou, Hui-Ren; Islam, Zahidul; Pestka, James J

    2003-03-01

    Since proinflammatory cytokine mRNA expression is induced within lymphoid tissue in vivo by the trichothecene vomitoxin (VT) in a rapid (1-2 h) and transient (4-8 h) fashion, it was hypothesized that mitogen-activated protein kinases (MAPKs) and transcription factors associated upstream with gene transcription of these cytokines are activated prior to or within these time windows. To test this hypothesis, mice were first treated with a single oral dose of VT and then analyzed for MAPK phosphorylation in the spleen. As little as 1 mg/kg of VT induced JNK 1/2, ERK 1/2, and p38 phosphorylation with maximal effects being observed at 5 to 100 mg/kg of VT. VT transiently induced JNK and p38 phosphorylation over a 60-min time period with peak effects being observed at 15 and 30 min, respectively. In contrast, ERK remained phosphorylated from 15 to 120 min. Next, the binding of activating protein 1 (AP-1), CCAAT enhancer-binding protein (C/EBP), CRE-binding protein (CREB), and nuclear factor-kappaB (NF-kappaB) was measured by electrophoretic mobility shift assay (EMSA) using four different consensus transcriptional control motifs at 0, 0.5, 1.5, 4, and 8 h after oral exposure to 25 mg/kg of VT. AP-1 binding activity was differentially elevated from 0.5 h to 8 h, whereas C/EBP binding was elevated only at 0.5 h. CREB binding decreased slightly at 0.5 h but gradually increased, reaching a maximum at 4 h. NF-kappaB binding was increased only slightly at 4 and 8 h. The specificities of AP-1, C/EBP, CREB, and NF-kappaB for relevant DNA motifs were verified by competition assays, using an excess of unlabeled consensus and mutant oligonucleotides. Supershift EMSAs and Western blot analysis identified specific VT-inducible DNA binding proteins for AP-1 (cJun, phospho c-jun, JunB, and JunD), C/EBP (C/EBPbeta), CREB (CREB-1 and ATF-2), and NF-kappaB (p50 and cRel). Finally, when the effects of oral VT exposure on proinflammatory gene expression were assessed at 3, 6, and 9 h

  4. RNA-binding protein Dnd1 inhibits microRNA access to target mRNA

    DEFF Research Database (Denmark)

    Kedde, Martijn; Strasser, Markus J; Boldajipour, Bijan

    2007-01-01

    MicroRNAs (miRNAs) are inhibitors of gene expression capable of controlling processes in normal development and cancer. In mammals, miRNAs use a seed sequence of 6-8 nucleotides (nt) to associate with 3' untranslated regions (3'UTRs) of mRNAs and inhibit their expression. Intriguingly, occasionally...... not only the miRNA-targeting site but also sequences in its vicinity are highly conserved throughout evolution. We therefore hypothesized that conserved regions in mRNAs may serve as docking platforms for modulators of miRNA activity. Here we demonstrate that the expression of dead end 1 (Dnd1......), an evolutionary conserved RNA-binding protein (RBP), counteracts the function of several miRNAs in human cells and in primordial germ cells of zebrafish by binding mRNAs and prohibiting miRNAs from associating with their target sites. These effects of Dnd1 are mediated through uridine-rich regions present...

  5. Women have higher protein content of beta-oxidation enzymes in skeletal muscle than men.

    Directory of Open Access Journals (Sweden)

    Amy C Maher

    Full Text Available It is well recognized that compared with men, women have better ultra-endurance capacity, oxidize more fat during endurance exercise, and are more resistant to fat oxidation defects i.e. diet-induced insulin resistance. Several groups have shown that the mRNA and protein transcribed and translated from genes related to transport of fatty acids into the muscle are greater in women than men; however, the mechanism(s for the observed sex differences in fat oxidation remains to be determined. Muscle biopsies from the vastus lateralis were obtained from moderately active men (N=12 and women (N=11 at rest to examine mRNA and protein content of genes involved in lipid oxidation. Our results show that women have significantly higher protein content for tri-functional protein alpha (TFPalpha, very long chain acyl-CoA dehydrogenase (VLCAD, and medium chain acyl-CoA dehydrogenase (MCAD (P<0.05. There was no significant sex difference in the expression of short-chain hydroxyacyl-CoA dehydrogenase (SCHAD, or peroxisome proliferator activated receptor alpha (PPARalpha, or PPARgamma, genes potentially involved in the transcriptional regulation of lipid metabolism. In conclusion, women have more protein content of the major enzymes involved in long and medium chain fatty acid oxidation which could account for the observed differences in fat oxidation during exercise.

  6. Synthesis of a metal binding protein designed on the alpha/beta scaffold of charybdotoxin.

    Science.gov (United States)

    Pierret, B; Virelizier, H; Vita, C

    1995-12-01

    The alpha/beta scaffold of the scorpion toxin charybdotoxin has been used for the engineering of a metal binding site. Nine substitutions, including three histidines as metal ligands, have been introduced into the original toxin sequence. The newly designed sequence, 37 amino acids long, has been assembled by solid-phase synthesis and HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) coupling of Fmoc-protected amino acids. Formation of the three disulfide bonds occurred efficiently and rapidly in the presence of glutathione, and this post-synthesis modification has facilitated the purification task enormously. The process of synthesis and purification was performed in less than a week with an overall 10.2% yield. Circular dichroism analysis showed that the newly designed protein is folded in a alpha/beta structure, similarly to the parent toxin. Electronic absorption spectroscopy, circular dichroism and gel filtration experiments have been used to show that Cu2+ and Zn2+ ions bind with high affinity to the newly engineered protein. These results demonstrate that the alpha/beta fold, common to all scorpion toxins, is a very versatile basic structure, tolerant for substitutions and able to present new sequences in a predetermined conformation. The chemical approach is shown to be effective, rapid and practical for the production of novel designed small proteins.

  7. The Microtubule-Associated Protein Tau and Its Relevance for Pancreatic Beta Cells.

    Science.gov (United States)

    Maj, Magdalena; Hoermann, Gregor; Rasul, Sazan; Base, Wolfgang; Wagner, Ludwig; Attems, Johannes

    2016-01-01

    Structural and biochemical alterations of the microtubule-associated protein tau (MAPT) are associated with degenerative disorders referred to as tauopathies. We have previously shown that MAPT is present in human islets of Langerhans, human insulinomas, and pancreatic beta-cell line models, with biophysical similarities to the pathological MAPT in the brain. Here, we further studied MAPT in pancreatic endocrine tissue to better understand the mechanisms that lead to functional dysregulation of pancreatic beta cells. We found upregulation of MAPT protein expression in human insulinomas when compared to human pancreatic islets of Langerhans and an imbalance between MAPT isoforms in insulinomas tissue. We cloned one 3-repeat domain MAPT and transduced this into a beta-cell derived rodent cell line Rin-5F. Proliferation experiments showed higher growth rates and metabolic activities of cells overexpressing MAPT protein. We observed that a MAPT overexpressing cell line demonstrates altered insulin transcription, translation, and insulin secretion rates. We found the relative insulin secretion rates were significantly decreased in a MAPT overexpressing cell line and these findings could be confirmed using partial MAPT knock-down cell lines. Our findings support that MAPT may play an important role in insulin granule trafficking and indicate the importance of balanced MAPT phosphorylation and dephosphorylation for adequate insulin release.

  8. The Microtubule-Associated Protein Tau and Its Relevance for Pancreatic Beta Cells

    Directory of Open Access Journals (Sweden)

    Magdalena Maj

    2016-01-01

    Full Text Available Structural and biochemical alterations of the microtubule-associated protein tau (MAPT are associated with degenerative disorders referred to as tauopathies. We have previously shown that MAPT is present in human islets of Langerhans, human insulinomas, and pancreatic beta-cell line models, with biophysical similarities to the pathological MAPT in the brain. Here, we further studied MAPT in pancreatic endocrine tissue to better understand the mechanisms that lead to functional dysregulation of pancreatic beta cells. We found upregulation of MAPT protein expression in human insulinomas when compared to human pancreatic islets of Langerhans and an imbalance between MAPT isoforms in insulinomas tissue. We cloned one 3-repeat domain MAPT and transduced this into a beta-cell derived rodent cell line Rin-5F. Proliferation experiments showed higher growth rates and metabolic activities of cells overexpressing MAPT protein. We observed that a MAPT overexpressing cell line demonstrates altered insulin transcription, translation, and insulin secretion rates. We found the relative insulin secretion rates were significantly decreased in a MAPT overexpressing cell line and these findings could be confirmed using partial MAPT knock-down cell lines. Our findings support that MAPT may play an important role in insulin granule trafficking and indicate the importance of balanced MAPT phosphorylation and dephosphorylation for adequate insulin release.

  9. Roles for transforming growth factor beta superfamily proteins in early folliculogenesis.

    Science.gov (United States)

    Trombly, Daniel J; Woodruff, Teresa K; Mayo, Kelly E

    2009-01-01

    Primordial follicle formation and the subsequent transition of follicles to the primary and secondary stages encompass the early events during folliculogenesis in mammals. These processes establish the ovarian follicle pool and prime follicles for entry into subsequent growth phases during the reproductive cycle. Perturbations during follicle formation can affect the size of the primordial follicle pool significantly, and alterations in follicle transition can cause follicles to arrest at immature stages or result in premature depletion of the follicle reserve. Determining the molecular events that regulate primordial follicle formation and early follicle growth may lead to the development of new fertility treatments. Over the last decade, many of the growth factors and signaling proteins that mediate the early stages of folliculogenesis have been identified using mouse genetic models, in vivo injection studies, and ex vivo organ culture approaches. These studies reveal important roles for the transforming growth factor beta (TGF-beta) superfamily of proteins in the ovary. This article reviews these roles for TGF-beta family proteins and focuses in particular on work from our laboratories on the functions of activin in early folliculogenesis.

  10. Nicotinic stimulation modulates tyrosine hydroxylase mRNA half-life and protein binding to the 3'UTR in a manner that requires transcription.

    Science.gov (United States)

    Roe, David F; Craviso, Gale L; Waymire, Jack C

    2004-01-05

    Tyrosine hydroxylase (TH) expression increases in adrenal chromaffin cells treated with the nicotinic agonist, dimethylphenylpiperazinium (DMPP; 1 microM). We are using this response as a model of the changes in TH level that occur during increased cholinergic neural activity. Here we report a 4-fold increase in TH mRNA half-life in DMPP-treated cells chromaffin cells that is apparent when using a pulse-chase analysis to measure TH mRNA half-life. No increase is apparent using actinomycin D to measure half-life, indicating a requirement for ongoing transcription. Characterization of protein binding to the TH 3'UTR responsible for stabilization using labeled TH 3'UTR probes and electro-mobility shift assays shows the presence of two complexes both of which are increased by DMPP-treatment. The faster migrating complex (FMC) increases 2.5-fold and the slower migrating complex (SMC) increases 1.5-fold. Both changes are prevented by actinomycin D. Characterization of the protein binding to the TH UTR probes indicates SMC is disrupted by polyribonucleotides, poly (A) and poly (U), while binding to FMC is reduced by poly (CU). Separation of UV crosslinked RNA-protein complexes on SDS polyacrylamide gels shows FMC to contain a single protein whereas SMC contains three proteins. Northwesterns yielded similar results. Comparison of DMPP-induced protein binding with the poly C binding protein (PCBP) involved in hypoxia induced rat PC12 TH mRNA stability indicates none of the bovine UTR binding proteins are the PCBP. Thus, nicotinic stimulation produces a transcription-dependent increase in TH mRNA half-life that is mediated by previously unrecognized TH mRNA binding proteins.

  11. Role of alpha-hemoglobin-stabilizing protein in normal erythropoiesis and beta-thalassemia.

    Science.gov (United States)

    Weiss, Mitchell J; Zhou, Suiping; Feng, Liang; Gell, David A; Mackay, Joel P; Shi, Yigong; Gow, Andrew J

    2005-01-01

    Hemoglobin (Hb) synthesis is coordinated by homeostatic mechanisms to limit the accumulation of free alpha or beta subunits, which are cytotoxic. Alpha hemoglobin-stabilizing protein (AHSP) is an abundant erythroid protein that specifically binds free alphaHb, stabilizes its structure, and limits its ability to participate in chemical reactions that generate reactive oxygen species. Gene ablation studies in mice demonstrate that AHSP is required for normal erythropoiesis. AHSP-null erythrocytes are short-lived, contain Hb precipitates, and exhibit signs of oxidative damage. Loss of AHSP exacerbates beta-thalassemia in mice, indicating that altered AHSP expression or function could modify thalassemia phenotypes in humans, a topic that is beginning to be explored in clinical studies. We used biochemical, spectroscopic, and crystallographic methods to examine how AHSP stabilizes alphaHb. AHSP binds the G and H helices of alphaHb on a surface that largely overlaps with the alpha1-beta1 interface of HbA. This result explains previous findings that betaHb can competitively displace AHSP from alphaHb to form HbA tetramer. Remarkably, binding of AHSP to oxygenated alphaHb induces dramatic conformational changes and converts the heme-bound iron to an oxidized hemichrome state in which all six coordinate positions are occupied. This structure limits the reactivity of heme iron, providing a mechanism by which AHSP stabilizes alphaHb. These findings suggest a biochemical pathway through which AHSP might participate in normal Hb synthesis and modulate the severity of thalassemias. Moreover, understanding how AHSP stabilizes alphaHb provides a theoretical basis for new strategies to inhibit the damaging effects of free alphaHb that accumulates in beta-thalassemia.

  12. Beta-keratins of turtle shell are glycine-proline-tyrosine rich proteins similar to those of crocodilians and birds.

    Science.gov (United States)

    Dalla Valle, Luisa; Nardi, Alessia; Toni, Mattia; Emera, Deena; Alibardi, Lorenzo

    2009-02-01

    This study presents, for the first time, sequences of five beta-keratin cDNAs from turtle epidermis obtained by means of 5'- and 3'-rapid amplification of cDNA ends (RACE) analyses. The deduced amino acid sequences correspond to distinct glycine-proline-serine-tyrosine rich proteins containing 122-174 amino acids. In situ hybridization shows that beta-keratin mRNAs are expressed in cells of the differentiating beta-layers of the shell scutes. Southern blotting analysis reveals that turtle beta-keratins belong to a well-conserved multigene family. This result was confirmed by the amplification and sequencing of 13 genomic fragments corresponding to beta-keratin genes. Like snake, crocodile and avian beta-keratin genes, turtle beta-keratins contain an intron that interrupts the 5'-untranslated region. The length of the intron is variable, ranging from 0.35 to 1.00 kb. One of the sequences obtained from genomic amplifications corresponds to one of the five sequences obtained from cDNA cloning; thus, sequences of a total of 17 turtle beta-keratins were determined in the present study. The predicted molecular weight of the 17 different deduced proteins range from 11.9 to 17.0 kDa with a predicted isoelectric point of 6.8-8.4; therefore, they are neutral to basic proteins. A central region rich in proline and with beta-strand conformation shows high conservation with other reptilian and avian beta-keratins, and it is likely involved in their polymerization. Glycine repeat regions, often containing tyrosine, are localized toward the C-terminus. Phylogenetic analysis shows that turtle beta-keratins are more similar to crocodilian and avian beta-keratins than to those of lizards and snakes.

  13. Stage-specific and species-specific differences in the production of the mRNA and protein for the filarial nematode secreted product, ES-62.

    Science.gov (United States)

    Stepek, G; Houston, K M; Goodridge, H S; Devaney, E; Harnett, W

    2004-01-01

    Previous studies have shown that the secreted phosphorylcholine-containing glycoprotein of filarial nematodes, ES-62, is only present in the post-infective life-cycle stages, but that the mRNA is transcribed throughout the worm's life-cycle. The aim of this current study was to investigate whether the presence or absence of protein expression simply reflects differences in mRNA abundance. To this end, we investigated the relative abundance of ES-62 using TaqMan real time RT-PCR, in different life-cycle stages of 2 model filarial nematode parasites, Acanthocheilonema viteae and Brugia pahangi. For B. pahangi, microfilariae, infective larvae and adult worms were each found to have approximately similar levels of ES-62 mRNA. However, the corresponding stages of A. viteae differed greatly from each other with a pattern of increased mRNA production with maturation. As a rule A. viteae had higher levels of ES-62 mRNA than B. pahangi, and this was particularly noticeable in the adult stage where the difference was approximately 3500-fold higher. However, this significant difference in mRNA abundance was not reflected in the quantity of ES-62 protein secreted by the adult worms of each species, as A. viteae only secreted approximately 3 times as much ES-62 as B. pahangi. Thus, overall, the results obtained from this study indicate that ES-62 protein production does not solely reflect mRNA levels, and also suggest that the 2 nematodes may employ different mechanisms for regulating protein production.

  14. Curcumin inhibits beta-amyloid protein 40/42 expression in the brain in a concentration-and time-dependent manner

    Institute of Scientific and Technical Information of China (English)

    Xiong Zhang; Lu Si; Xiaodong Shi; Wenke Yin; Yu Li

    2010-01-01

    Several studies have demonstrated that the amount of beta-amyloid(Aβ)protein in the brain can be lowered by down-regulating Aβ production,promoting Aβ degradation,reducing Aβ oligomerization or deposition,thereby alleviating symptoms of Alzheimer's disease.Curcumin has been known to be a peroxisome proliferator activated receptor gamma(PPARy)agonist and can obviously inhibit Aβ production and oligomerization.This study investigated the effects of curcumin on the β-site APP cleaving enzyme 1(BACE1)activity and PPARy expression in human neuroblastoma SH-SY5Y cells,and validated the inhibitory effects of curcumin on Aβ40/42 expression in the brain.Results revealed that PPARy mRNA and protein expression in the human neuroblastoma SH-SY5Y cells significantly increased with increasing curcumin concentration and time course(P < 0.05);BACE1 mRNA and protein expression and Aβ40/42 production significantly decreased with increasing curcumin concentration and time course(P < 0.05).The changes in PPARY and BACE1expression during Aβ production could be reversed by the PPARy antagonist GW9662.These findings indicate that curcumin reduced Aβ production by activating PPARy expression and inhibiting BACE1 expression in a concentration-and time-dependent manner.

  15. Have We Overlooked the Importance of Serine/Threonine Protein Phosphatases in Pancreatic Beta-Cells? Role Played by Protein Phosphatase 2A in Insulin Secretion

    Directory of Open Access Journals (Sweden)

    Esser V

    2005-07-01

    Full Text Available Genetic predisposition and environmental influences insidiously converge to cause glucose intolerance and hyperglycemia. Beta-cell compensates by secreting more insulin and when it fails, overt diabetes mellitus ensues. The need to understand the mechanisms involved in insulin secretion cannot be stressed enough. Phosphorylation of proteins plays an important role in regulating insulin secretion. In order to understand how a particular cellular process is regulated by protein phosphorylation the nature of the protein kinases and protein phosphatases involved and the mechanisms that determine when and where these enzymes are active should be investigated. While the actions of protein kinases have been intensely studied within the beta-cell, less emphasis has been placed on protein phosphatases even though they play an important regulatory role. This review focuses on the importance of protein phosphatase 2A in insulin secretion. Most of the present knowledge on protein phosphatase 2A originates from protein phosphatase inhibitor studies on islets and beta-cell lines. The ability of protein phosphatase 2A to change its activity in the presence of glucose and inhibitors provides clues to its role in regulating insulin secretion. An aggressive approach to elucidate the substrates and mechanisms of action of protein phosphatases is crucial to the understanding of phosphorylation events within the beta-cell. Characterizing protein phosphatase 2A within the beta-cell will certainly help us in understanding the mechanisms involved in insulin secretion and provide valuable information for drug development.

  16. Improved Risk Stratification in Pediatric Septic Shock Using Both Protein and mRNA Biomarkers. PERSEVERE-XP.

    Science.gov (United States)

    Wong, Hector R; Cvijanovich, Natalie Z; Anas, Nick; Allen, Geoffrey L; Thomas, Neal J; Bigham, Michael T; Weiss, Scott L; Fitzgerald, Julie C; Checchia, Paul A; Meyer, Keith; Quasney, Michael; Hall, Mark; Gedeit, Rainer; Freishtat, Robert J; Nowak, Jeffrey; Raj, Shekhar S; Gertz, Shira; Grunwell, Jocelyn R; Lindsell, Christopher J

    2017-08-15

    We previously derived and validated the Pediatric Sepsis Biomarker Risk Model (PERSEVERE) to estimate baseline mortality risk in children with septic shock. The PERSEVERE biomarkers are serum proteins selected from among the proteins directly related to 80 mortality risk assessment genes. The initial approach to selecting the PERSEVERE biomarkers left 68 genes unconsidered. To determine if the 68 previously unconsidered genes can improve upon the performance of PERSEVERE and to provide biological information regarding the pathophysiology of septic shock. We reduced the number of variables by determining the biological linkage of the 68 previously unconsidered genes. The genes identified through variable reduction were combined with the PERSEVERE-based mortality probability to derive a risk stratification model for 28-day mortality using classification and regression tree methodology (n = 307). The derived tree, PERSEVERE-XP, was then tested in a separate cohort (n = 77). Variable reduction revealed a network consisting of 18 mortality risk assessment genes related to tumor protein 53 (TP53). In the derivation cohort, PERSEVERE-XP had an area under the receiver operating characteristic curve (AUC) of 0.90 (95% confidence interval, 0.85-0.95) for differentiating between survivors and nonsurvivors. In the test cohort, the AUC was 0.96 (95% confidence interval, 0.91-1.0). The AUC of PERSEVERE-XP was superior to that of PERSEVERE. PERSEVERE-XP combines protein and mRNA biomarkers to provide mortality risk stratification with possible clinical utility. PERSEVERE-XP significantly improves on PERSEVERE and suggests a role for TP53-related cellular division, repair, and metabolism in the pathophysiology of septic shock.

  17. Mechanism of cyst specific protein 21 mRNA induction during Acanthamoeba differentiation.

    Science.gov (United States)

    Chen, Li; Orfeo, Tom; Gilmartin, Greg; Bateman, Erik

    2004-04-01

    The Acanthamoeba cyst specific protein 21 (CSP21) gene is tightly repressed in growing cells and highly induced early during differentiation into a dormant cyst. This increase is mediated by the rate of transcription of the CSP21 gene as determined by nuclear run-on assays. The promoter region of the CSP21 gene was analyzed by transcript start site mapping and in vitro transcription of wild-type or mutant templates, using extracts from growing cells. A sequence located 3' to a modified TATA box completely inhibits transcription and removal of this region permits robust transcription utilizing a start site approximately 35 base pairs downstream of the TATA box. Sequences 5' to the TATA box had no effect on transcription, suggesting that anti-repression is the only mechanism required for CSP21 induction. Fractionation of nuclear extracts yielded a fraction capable of transcription from the CSP21 promoter, and a fraction containing a promoter-specific repressing activity. Anti-repression may thus be a major mechanism regulating differentiation or maintenance of the proliferative cycle in Acanthamoeba.

  18. Expression of Heat Shock Protein 70 mRNA in Epithelial Cells of Human Lens

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Objective:To try to find out the pathogenesis of the cataract , effects of beat and oxidative stresson heat shock proteins of tissue cultured human lens epithelial cells (LEC-B3) were investigate& Methods:Cells were exposed to heat shock (45℃) and oxidative stress(5OmMH2O2 for 30 min, and then allowed to recoverat different intervals (Oh, 2h, 4h, 6h, 16h, 24h) in physiological medium Reverse transcription polymerasechain reaction (RT-PCR) were used to determined the level of HSP70. Results: HSPs existed in both physiologicaland stressful situation. The level of HSP7OmRNA increased 2h later after both stresses. The expression of HSP70got to the summit during 2h to 6h in each group. Subsequently it decreased gradually in each group, maintaininga high level at 16h. Conclusion: HSP70 exists in lens epithelial cells and can be induced after stress. Thedata suggested it may play an important protective role in lens epithelial cells in respond to cellular stress.

  19. Zygote arrest 1, nucleoplasmin 2, and developmentally associated protein 3 mRNA profiles throughout porcine embryo development in vitro.

    Science.gov (United States)

    Wasielak, M; Więsak, T; Bogacka, I; Jalali, B Moza; Bogacki, M

    2016-12-01

    Maternal effect genes (MEGs) are expressed in oocytes and embryos and play an important role in activation of the embryonic genome. An abnormality in the expression of these genes may lead to arrest of embryonic cleavage or to altered transcription of factors responsible for further embryonic development. In vitro-produced porcine embryos have a lower developmental potential than embryos produced in vivo. We hypothesized that in vitro embryo culture conditions have an effect on the expression of MEGs at various developmental stages, which may affect their developmental potential. Here, using real-time polymerase chain reaction, we examined mRNA profiles of the MEGs, zygote arrest 1 (ZAR-1), nucleoplasmin 2 (NPM2), and developmentally associated pluripotency protein 3 (DPPA3), in porcine oocytes and embryos produced in vitro and in vivo. Further, we evaluated the effect of the combined addition of EGF, interleukin 1β, and leukemia inhibitory factor to the porcine in vitro embryo production system on mRNA profiles of selected MEGs. Finally, we studied localization of the MEG protein products in in vitro-obtained oocytes and embryos using confocal microscopy. We found that the ZAR-1 mRNA profile differed throughout in vitro and in vivo embryo development. In the embryos produced in vitro, the decrease in ZAR-1 mRNA levels was observed at the 2-cell stage, whereas in in vivo embryos, ZAR-1 mRNA levels declined significantly starting at the 4-cell stage (P vitro culture conditions affected transiently also DPPA3 mRNA levels at the 4-cell stage (P vitro and in vivo embryo development. The ZAR-1 and DPPA3 proteins were localized in the cytoplasm of the oocytes and embryos, whereas the NPM2 protein was found both in the cytoplasm and in the nucleus. All proteins were expressed until blastocyst stage. The addition of EGF and cytokines to the culture medium decreased DPPA3 mRNA levels in 8-cell embryos (P < 0.05). This study indicated that IVC conditions

  20. Calcium ionophore A23187 specifically decreases the secretion of beta-secretase cleaved amyloid precursor protein during apoptosis in primary rat cortical cultures

    DEFF Research Database (Denmark)

    Sennvik, K; Benedikz, Eirikur; Fastbom, J;

    2001-01-01

    Alzheimer's disease (AD) is characterized by the degeneration and loss of neurons, intracellular neurofibrillary tangles and the accumulation of extracellular senile plaques consisting mainly of beta-amyloid (A beta). A beta is generated from the amyloid precursor protein (APP) by sequential beta...

  1. Toxicological effects of nickel chloride on the cytokine mRNA expression and protein levels in intestinal mucosal immunity of broilers.

    Science.gov (United States)

    Wu, Bangyuan; Cui, Hengmin; Peng, Xi; Fang, Jing; Zuo, Zhicai; Deng, Junliang; Wang, Xun; Huang, Jianying

    2015-11-01

    The purpose of this study was to examine the toxicological effects of nickel chloride (NiCl2 ; 300, 600, and 900 mg kg(-1) diet) on the cytokine mRNA expression and protein levels in the intestinal mucosa and cecal tonsil, and on the ileac and cecal tonsil T cells in broilers by the methods of qRT-PCR, flow cytometry and ELISA for 42 days. Results showed that the IL-2, IL-6, IL-10, IL-17, IFN-γ, and TNF-α (LITAF) cytokine mRNA expression and protein levels were lower (P mucosal immunity.

  2. Integrated analysis of global mRNA and protein expression data in HEK293 cells overexpressing PRL-1.

    Directory of Open Access Journals (Sweden)

    Carmen M Dumaual

    Full Text Available BACKGROUND: The protein tyrosine phosphatase PRL-1 represents a putative oncogene with wide-ranging cellular effects. Overexpression of PRL-1 can promote cell proliferation, survival, migration, invasion, and metastasis, but the underlying mechanisms by which it influences these processes remain poorly understood. METHODOLOGY: To increase our comprehension of PRL-1 mediated signaling events, we employed transcriptional profiling (DNA microarray and proteomics (mass spectrometry to perform a thorough characterization of the global molecular changes in gene expression that occur in response to stable PRL-1 overexpression in a relevant model system (HEK293. PRINCIPAL FINDINGS: Overexpression of PRL-1 led to several significant changes in the mRNA and protein expression profiles of HEK293 cells. The differentially expressed gene set was highly enriched in genes involved in cytoskeletal remodeling, integrin-mediated cell-matrix adhesion, and RNA recognition and splicing. In particular, members of the Rho signaling pathway and molecules that converge on this pathway were heavily influenced by PRL-1 overexpression, supporting observations from previous studies that link PRL-1 to the Rho GTPase signaling network. In addition, several genes not previously associated with PRL-1 were found to be significantly altered by its expression. Most notable among these were Filamin A, RhoGDIα, SPARC, hnRNPH2, and PRDX2. CONCLUSIONS AND SIGNIFICANCE: This systems-level approach sheds new light on the molecular networks underlying PRL-1 action and presents several novel directions for future, hypothesis-based studies.

  3. Data supporting the effects of lysozyme on mRNA and protein expression in a colonic epithelial scratch wound model

    Directory of Open Access Journals (Sweden)

    Sarah K. Abey

    2017-04-01

    Full Text Available Colonic epithelial health is implicated in a host of gastrointestinal (GI diseases and disorders. Lysozyme is suspected to play a role in the ability of the epithelium to recover from injury (Abey et al., in press; Gallo, 2012; Rubio, 2014 [1–3]. Disrupted repair mechanisms may lead to delayed or ineffective recovery and disruptions to epithelial biology resulting in GI symptoms and altered barrier function (Peterson and Artis, 2014 [4]. The effect of lysozyme on the transcriptomic and proteomic profile of healthy colonic epithelial cells was investigated. Epithelial cells in culture were scratch wounded and treated with lysozyme. mRNA and protein profiles were simultaneously quantified in the same sample using a digital counting technology. Gene and protein expressions altered by the presence or absence of lysozyme are described in this article. Extensive statistical and bioinformatic analysis, and interpretation of the results can be found in “Lysozyme association with circulating RNA, extracellular vesicles, and chronic stress” (Abey et al., in press [1].

  4. Nanoscale organization of {beta}{sub 2}-adrenergic receptor-Venus fusion protein domains on the surface of mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Vobornik, Dusan; Rouleau, Yanouchka; Haley, Jennifer [Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, ON, Canada K1A 0R6 (Canada); Bani-Yaghoub, Mahmud [Institute for Biological Sciences, National Research Council Canada, Ottawa, ON, Canada K1A 0R6 (Canada); Taylor, Rod [Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, ON, Canada K1A 0R6 (Canada); Johnston, Linda J., E-mail: Linda.Johnston@nrc-cnrc.gc.ca [Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, ON, Canada K1A 0R6 (Canada); Pezacki, John Paul, E-mail: John.Pezacki@nrc-cnrc.gc.ca [Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, ON, Canada K1A 0R6 (Canada)

    2009-04-24

    Adrenergic receptors are a key component of nanoscale multiprotein complexes that are responsible for controlling the beat rate in a mammalian heart. We demonstrate the ability of near-field scanning optical microscopy (NSOM) to visualize {beta}{sub 2}-adrenergic receptors ({beta}{sub 2}AR) fused to the GFP analogue Venus at the nanoscale on HEK293 cells. The expression of the {beta}{sub 2}AR-Venus fusion protein was tightly controlled using a tetracycline-induced promoter. Both the size and density of the observed nanoscale domains are dependent on the level of induction and thus the level of protein expression. At concentrations between 100 and 700 ng/ml of inducer doxycycline, the size of domains containing the {beta}{sub 2}AR-Venus fusion protein appears to remain roughly constant, but the number of domains per cell increase. At 700 ng/ml doxycycline the functional receptors are organized into domains with an average diameter of 150 nm with a density similar to that observed for the native protein on primary murine cells. By contrast, larger micron-sized domains of {beta}{sub 2}AR are observed in the membrane of the HEK293 cells that stably overexpress {beta}{sub 2}AR-GFP and {beta}{sub 2}AR-eYFP. We conclude that precise chemical control of gene expression is highly advantageous for the use {beta}{sub 2}AR-Venus fusion proteins as models for {beta}{sub 2}AR function. These observations are critical for designing future cell models and assays based on {beta}{sub 2}AR, since the receptor biology is consistent with a relatively low density of nanoscale receptor domains.

  5. Beta-site amyloid precursor protein cleaving enzyme 1 levels become elevated in neurons around amyloid plaques: implications for Alzheimer's disease pathogenesis.

    Science.gov (United States)

    Zhao, Jie; Fu, Yifan; Yasvoina, Marina; Shao, Peizhen; Hitt, Brian; O'Connor, Tracy; Logan, Sreemathi; Maus, Erika; Citron, Martin; Berry, Robert; Binder, Lester; Vassar, Robert

    2007-04-04

    Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) (beta-secretase) initiates generation of beta-amyloid (Abeta), which plays an early role in Alzheimer's disease (AD). BACE1 levels are increased in postmortem AD brain, suggesting BACE1 elevation promotes Abeta production and AD. Alternatively, the BACE1 increase may be an epiphenomenon of late-stage AD. To distinguish between these possibilities, we analyzed BACE1 elevation using a highly specific BACE1 antibody, BACE-Cat1, made in BACE1-/- mice, which mount a robust anti-BACE1 immune response. Previous BACE1 immunohistochemical studies lack consistent results because typical BACE1 antibodies produce nonspecific background, but BACE-Cat1 immunolabels BACE1 only. BACE1 elevation was recapitulated in two amyloid precursor protein (APP) transgenic mouse lines. 5XFAD mice form amyloid plaques at young ages and exhibit neuron loss. In contrast, Tg2576 form plaques at a more advanced age and do not show cell death. These two mouse lines allow differentiation between early Abeta-induced events and late phenomena related to neuron death. BACE1 levels became elevated in parallel with amyloid burden in each APP transgenic, starting early in 5XFAD and late in Tg2576. The increase in BACE1 protein occurred without any change in BACE1 mRNA level, indicating a posttranscriptional mechanism. In APP transgenic and AD brains, high BACE1 levels were observed in an annulus around Abeta42-positive plaque cores and colocalized with neuronal proteins. These results demonstrate that amyloid plaques induce BACE1 in surrounding neurons at early stages of pathology before neuron death occurs. We conclude that BACE1 elevation is most likely triggered by the amyloid pathway and may drive a positive-feedback loop in AD.

  6. Structural basis for the recognition of cellular mRNA export factor REF by herpes viral proteins HSV-1 ICP27 and HVS ORF57.

    Directory of Open Access Journals (Sweden)

    Richard B Tunnicliffe

    2011-01-01

    Full Text Available The herpesvirus proteins HSV-1 ICP27 and HVS ORF57 promote viral mRNA export by utilizing the cellular mRNA export machinery. This function is triggered by binding to proteins of the transcription-export (TREX complex, in particular to REF/Aly which directs viral mRNA to the TAP/NFX1 pathway and, subsequently, to the nuclear pore for export to the cytoplasm. Here we have determined the structure of the REF-ICP27 interaction interface at atomic-resolution and provided a detailed comparison of the binding interfaces between ICP27, ORF57 and REF using solution-state NMR. Despite the absence of any obvious sequence similarity, both viral proteins bind on the same site of the folded RRM domain of REF, via short but specific recognition sites. The regions of ICP27 and ORF57 involved in binding by REF have been mapped as residues 104-112 and 103-120, respectively. We have identified the pattern of residues critical for REF/Aly recognition, common to both ICP27 and ORF57. The importance of the key amino acid residues within these binding sites was confirmed by site-directed mutagenesis. The functional significance of the ORF57-REF/Aly interaction was also probed using an ex vivo cytoplasmic viral mRNA accumulation assay and this revealed that mutants that reduce the protein-protein interaction dramatically decrease the ability of ORF57 to mediate the nuclear export of intronless viral mRNA. Together these data precisely map amino acid residues responsible for the direct interactions between viral adaptors and cellular REF/Aly and provide the first molecular details of how herpes viruses access the cellular mRNA export pathway.

  7. Age-related protein and mRNA expression of glutathione peroxidases (GPx and Hsp-70 in different regions of rat kidney with and without stressor

    Directory of Open Access Journals (Sweden)

    Noor Riyadh Thiab

    2016-04-01

    Full Text Available Small molecular weight oxygen free radical species (ROS involved in oxidative stress can cause damage to cellular macromolecules including proteins, DNA and lipids. One of the most important enzymes involved in ROS detoxification is glutathione peroxidase (GPx. Here we study the age-related expression of GPx isoenzymes in various parts of the rat kidney with and without exposure to external oxidative stress. These results are correlated to the age dependent changes in the expression of the chaperone, Hsp-70. Protein and mRNA expression of GPx1 and GPx4 was studied in different regions of the kidney in ageing rats in the presence and absence of the external stressor 0.2 mM H2O2. Protein levels were examined by Western blot analysis following detection with appropriate antibodies and mRNA levels were analysed by quantitative reverse transcription polymerase chain reaction (qRT-PCR using appropriate primer sequences. mRNA expression for the chaperone Hsp70 was investigated in parallel. After reaching a peak at maturity (12 weeks, GPx1 protein and mRNA levels decreased with age under both control and stress conditions, and were higher in the cortex than in the outer and inner medulla. GPx4 protein and mRNA levels showed few comparable age-related changes. By contrast with the observed age-related decrease in GPx1 expression, chaperone Hsp-70 mRNA expression greatly increased with age. These findings suggest that the age-related decline in GPx1 expression in the cortex may be partly offset by a reciprocal change in Hsp-70 expression. These results are consistent with the oxidative stress theory of ageing.

  8. Structural basis for the recognition of cellular mRNA export factor REF by herpes viral proteins HSV-1 ICP27 and HVS ORF57.

    Science.gov (United States)

    Tunnicliffe, Richard B; Hautbergue, Guillaume M; Kalra, Priti; Jackson, Brian R; Whitehouse, Adrian; Wilson, Stuart A; Golovanov, Alexander P

    2011-01-06

    The herpesvirus proteins HSV-1 ICP27 and HVS ORF57 promote viral mRNA export by utilizing the cellular mRNA export machinery. This function is triggered by binding to proteins of the transcription-export (TREX) complex, in particular to REF/Aly which directs viral mRNA to the TAP/NFX1 pathway and, subsequently, to the nuclear pore for export to the cytoplasm. Here we have determined the structure of the REF-ICP27 interaction interface at atomic-resolution and provided a detailed comparison of the binding interfaces between ICP27, ORF57 and REF using solution-state NMR. Despite the absence of any obvious sequence similarity, both viral proteins bind on the same site of the folded RRM domain of REF, via short but specific recognition sites. The regions of ICP27 and ORF57 involved in binding by REF have been mapped as residues 104-112 and 103-120, respectively. We have identified the pattern of residues critical for REF/Aly recognition, common to both ICP27 and ORF57. The importance of the key amino acid residues within these binding sites was confirmed by site-directed mutagenesis. The functional significance of the ORF57-REF/Aly interaction was also probed using an ex vivo cytoplasmic viral mRNA accumulation assay and this revealed that mutants that reduce the protein-protein interaction dramatically decrease the ability of ORF57 to mediate the nuclear export of intronless viral mRNA. Together these data precisely map amino acid residues responsible for the direct interactions between viral adaptors and cellular REF/Aly and provide the first molecular details of how herpes viruses access the cellular mRNA export pathway.

  9. PAB-1, a Caenorhabditis elegans poly(A-binding protein, regulates mRNA metabolism in germline by interacting with CGH-1 and CAR-1.

    Directory of Open Access Journals (Sweden)

    Sunhee Ko

    Full Text Available Poly(A-binding proteins are highly conserved among eukaryotes and regulate stability of mRNA and translation. Among C. elegans homologues, pab-1 mutants showed defects in germline mitotic proliferation. Unlike pab-1 mutants, pab-1 RNAi at every larval stage caused arrest of germline development at the following stage, indicating that pab-1 is required for the entire postembryonic germline development. This idea is supported by the observations that the mRNA level of pab-1 increased throughout postembryonic development and its protein expression was germline-enriched. PAB-1 localized to P granules and the cytoplasm in the germline. PAB-1 colocalized with CGH-1 and CAR-1 and affected their localization, suggesting that PAB-1 is a component of processing (P-bodies that interacts with them. The mRNA and protein levels of representative germline genes, rec-8, GLP-1, rme-2, and msp-152, were decreased after pab-1 RNAi. Although the mRNA level of msp-152 was increased in cgh-1 mutant, it was also significantly reduced by pab-1 RNAi. Our results suggest that PAB-1 positively regulates the mRNA levels of germline genes, which is likely facilitated by the interaction of PAB-1 with other P-body components, CGH-1 and CAR-1.

  10. PAB-1, a Caenorhabditis elegans poly(A)-binding protein, regulates mRNA metabolism in germline by interacting with CGH-1 and CAR-1.

    Science.gov (United States)

    Ko, Sunhee; Kawasaki, Ichiro; Shim, Yhong-Hee

    2013-01-01

    Poly(A)-binding proteins are highly conserved among eukaryotes and regulate stability of mRNA and translation. Among C. elegans homologues, pab-1 mutants showed defects in germline mitotic proliferation. Unlike pab-1 mutants, pab-1 RNAi at every larval stage caused arrest of germline development at the following stage, indicating that pab-1 is required for the entire postembryonic germline development. This idea is supported by the observations that the mRNA level of pab-1 increased throughout postembryonic development and its protein expression was germline-enriched. PAB-1 localized to P granules and the cytoplasm in the germline. PAB-1 colocalized with CGH-1 and CAR-1 and affected their localization, suggesting that PAB-1 is a component of processing (P)-bodies that interacts with them. The mRNA and protein levels of representative germline genes, rec-8, GLP-1, rme-2, and msp-152, were decreased after pab-1 RNAi. Although the mRNA level of msp-152 was increased in cgh-1 mutant, it was also significantly reduced by pab-1 RNAi. Our results suggest that PAB-1 positively regulates the mRNA levels of germline genes, which is likely facilitated by the interaction of PAB-1 with other P-body components, CGH-1 and CAR-1.

  11. Determinants of binding affinity and specificity for the interaction of TEM-1 and SME-1 beta-lactamase with beta-lactamase inhibitory protein.

    Science.gov (United States)

    Zhang, Zhen; Palzkill, Timothy

    2003-11-14

    The hydrolysis of beta-lactam antibiotics by class A beta-lactamases is a common cause of bacterial resistance to these agents. The beta-lactamase inhibitory protein (BLIP) is able to bind and inhibit several class A beta-lactamases, including TEM-1 beta-lactamase and SME-1 beta-lactamase. Although the TEM-1 and SME-1 enzymes share 33% amino acid sequence identity and a similar fold, they differ substantially in surface electrostatic properties and the conformation of a loop-helix region that BLIP binds. Alanine-scanning mutagenesis was performed to identify the residues on BLIP that contribute to its binding affinity for each of these enzymes. The results indicate that the sequence requirements for binding are similar for both enzymes with most of the binding free energy provided by two patches of aromatic residues on the surface of BLIP. Polar residues such as several serines in the interface do not make significant contributions to affinity for either enzyme. In addition, the specificity of binding is significantly altered by mutation of two charged residues, Glu73 and Lys74, that are buried in the structure of the TEM-1.BLIP complex as well as by residues located on two loops that insert into the active site pocket. Based on the results, a E73A/Y50A double mutant was constructed that exhibited a 220,000-fold change in binding specificity for the TEM-1 versus SME-1 enzymes.

  12. The interaction of the cellular export adaptor protein Aly/REF with ICP27 contributes to the efficiency of herpes simplex virus 1 mRNA export.

    Science.gov (United States)

    Tian, Xiaochen; Devi-Rao, Gayathri; Golovanov, Alexander P; Sandri-Goldin, Rozanne M

    2013-07-01

    Herpes simplex virus 1 (HSV-1) protein ICP27 enables viral mRNA export by accessing the cellular mRNA export receptor TAP/NXF, which guides mRNA through the nuclear pore complex. ICP27 binds viral mRNAs and interacts with TAP/NXF, providing a link to the cellular mRNA export pathway. ICP27 also interacts with the mRNA export adaptor protein Aly/REF, which binds cellular mRNAs and also interacts with TAP/NXF. Studies using small interfering RNA (siRNA) knockdown indicated that Aly/REF is not required for cellular mRNA export, and similar knockdown studies during HSV-1 infection led us to conclude that Aly/REF may be dispensable for viral RNA export. Recently, the structural basis of the interaction of ICP27 with Aly/REF was elucidated at atomic resolution, and it was shown that three ICP27 residues, W105, R107, and L108, interface with the RNA recognition motif (RRM) domain of Aly/REF. Here, to determine the role the interaction of ICP27 and Aly/REF plays during infection, these residues were mutated to alanine, and a recombinant virus, WRL-A, was constructed. Virus production was reduced about 10-fold during WRL-A infection, and export of ICP27 protein and most viral mRNAs was less efficient. We conclude that interaction of ICP27 with Aly/REF contributes to efficient viral mRNA export.

  13. mRNA and protein expression of FGF-1, FGF-2 and their receptors in the porcine umbilical cord during pregnancy.

    Directory of Open Access Journals (Sweden)

    R Rekawiecki

    2011-04-01

    Full Text Available The fibroblast growth factors (FGFs are multifunctional proteins that, among other roles, regulate structural reorganization of uterine and placental vascular bed during pregnancy. Thus, we analyzed mRNA and protein expression and immunohistochemical localization of FGF-1 and FGF-2, and their receptors (FGFR-1 and FGFR-2 in the developing umbilical cord (UC on days 40, 60, 75 and 90 of pregnancy and after the physiological delivery in the pig (day 114. qPCR analysis demonstrated an increase in FGF-1 and FGF-2 mRNA levels beginning on day 75 and on day 114 of pregnancy, respectively. In addition, significantly increased FGFR-1IIIc mRNA expression was also found on day 114. On the other hand, no significant changes in FGFR-2IIIb mRNA expression were observed. Western Blot analysis revealed a decrease in FGF-1 and FGFR-2 protein expression after day 40. Beside an increased protein expression of FGF-2 on day 60, no significant changes in FGFR-1 protein expression were detected. Immunohistochemical staining enabled detection of FGF-FGFR system, with different intensity of immunoreaction in endothelial and tunica media cells of the umbilical vessels and in allantoic duct and amniotic epithelium as well as in myofibroblasts. In conclusion, our results show that members of FGF-FGFR system are expressed specifically in UC structures. Furthermore their day of pregnancy-related expression suggest that they may be an important players during UC formation and development.

  14. Prediction of the beta-hairpins in proteins using support vector machine.

    Science.gov (United States)

    Hu, Xiu Zhen; Li, Qian Zhong

    2008-02-01

    By using of the composite vector with increment of diversity and scoring function to express the information of sequence, a support vector machine (SVM) algorithm for predicting beta-hairpin motifs is proposed. The prediction is done on a dataset of 3,088 non homologous proteins containing 6,027 beta-hairpins. The overall accuracy of prediction and Matthew's correlation coefficient are 79.9% and 0.59 for the independent testing dataset. In addition, a higher accuracy of 83.3% and Matthew's correlation coefficient of 0.67 in the independent testing dataset are obtained on a dataset previously used by Kumar et al. (Nuclic Acid Res 33:154-159). The performance of the method is also evaluated by predicting the beta-hairpins of in the CASP6 proteins, and the better results are obtained. Moreover, this method is used to predict four kinds of supersecondary structures. The overall accuracy of prediction is 64.5% for the independent testing dataset.

  15. Labelling of penicillin-binding proteins from Escherichia coli with photoreactive derivatives of. beta. -lactam antibiotics

    Energy Technology Data Exchange (ETDEWEB)

    Aran, V.; Rodriguez-Tebar, A.; Vazquez, D. (Universidad Autonoma de Madrid (Spain))

    1983-03-21

    The authors have synthesized a number of photoreactive radiolabelled ..beta..-lactams that react and form permanent covalent bonds with the penicillin-binding proteins (PBPs), since photoreactive ligand derivatives have been used to some extent for structural studies on membranes and other biological structures. Chemical and photochemical labelling of a receptor by its ligand are important techniques to elucidate the nature of the ligand-receptor interaction, and for identification and characterization of receptors. They have synthesized two ..beta..-lactam derivatives each containing two different photoreactive moieties. One of them is an aryl azido compound, widely known as a photoreactive reagent for labelling studies, whereas the other one contains a nitroguaiacol derived group used in photochemical studies with other biological materials.

  16. The addition of beta-hydroxy-beta-methylbutyrate and isomaltulose to whey protein improves recovery from highly demanding resistance exercise.

    Science.gov (United States)

    Kraemer, William J; Hooper, David R; Szivak, Tunde K; Kupchak, Brian R; Dunn-Lewis, Courtenay; Comstock, Brett A; Flanagan, Shawn D; Looney, David P; Sterczala, Adam J; DuPont, William H; Pryor, J Luke; Luk, Hiu-Ying; Maladoungdock, Jesse; McDermott, Danielle; Volek, Jeff S; Maresh, Carl M

    2015-01-01

    This study evaluated whether a combination of whey protein (WP), calcium beta-hydroxy-beta-methylbutyrate (HMB), and carbohydrate exert additive effects on recovery from highly demanding resistance exercise. Thirteen resistance-trained men (age: 22.6 ± 3.9 years; height: 175.3 ± 12.2 cm; weight: 86.2 ± 9.8 kg) completed a double-blinded, counterbalanced, within-group study. Subjects ingested EAS Recovery Protein (RP; EAS Sports Nutrition/Abbott Laboratories, Columbus, OH) or WP twice daily for 2 weeks prior to, during, and for 2 days following 3 consecutive days of intense resistance exercise. The workout sequence included heavy resistance exercise (day 1) and metabolic resistance exercise (days 2 and 3). The subjects performed no physical activity during day 4 (+24 hours) and day 5 (+48 hours), where recovery testing was performed. Before, during, and following the 3 workouts, treatment outcomes were evaluated using blood-based muscle damage markers and hormones, perceptual measures of muscle soreness, and countermovement jump performance. Creatine kinase was lower for the RP treatment on day 2 (RP: 166.9 ± 56.4 vs WP: 307.1 ± 125.2 IU · L(-1), p ≤ 0.05), day 4 (RP: 232.5 ± 67.4 vs WP: 432.6 ± 223.3 IU · L(-1), p ≤ 0.05), and day 5 (RP: 176.1 ± 38.7 vs 264.5 ± 120.9 IU · L(-1), p ≤ 0.05). Interleukin-6 was lower for the RP treatment on day 4 (RP: 1.2 ± 0.2 vs WP: 1.6 ± 0.6 pg · ml(-1), p ≤ 0.05) and day 5 (RP: 1.1 ± 0.2 vs WP: 1.6 ± 0.4 pg · ml(-1), p ≤ 0.05). Muscle soreness was lower for RP treatment on day 4 (RP: 2.0 ± 0.7 vs WP: 2.8 ± 1.1 cm, p ≤ 0.05). Vertical jump power was higher for the RP treatment on day 4 (RP: 5983.2 ± 624 vs WP 5303.9 ± 641.7 W, p ≤ 0.05) and day 5 (RP: 5792.5 ± 595.4 vs WP: 5200.4 ± 501 W, p ≤ 0.05). Our findings suggest that during times of intense conditioning, the recovery benefits of WP are enhanced with the addition of HMB and a slow-release carbohydrate. We

  17. mRNA decay proteins are targeted to poly(A+ RNA and dsRNA-containing cytoplasmic foci that resemble P-bodies in Entamoeba histolytica.

    Directory of Open Access Journals (Sweden)

    Itzel López-Rosas

    Full Text Available In higher eukaryotes, mRNA degradation and RNA-based gene silencing occur in cytoplasmic foci referred to as processing bodies (P-bodies. In protozoan parasites, the presence of P-bodies and their putative role in mRNA decay have yet to be comprehensively addressed. Identification of P-bodies might provide information on how mRNA degradation machineries evolved in lower eukaryotes. Here, we used immunofluorescence and confocal microscopy assays to investigate the cellular localization of mRNA degradation proteins in the human intestinal parasite Entamoeba histolytica and found evidence of the existence of P-bodies. Two mRNA decay factors, namely the EhXRN2 exoribonuclease and the EhDCP2 decapping enzyme, were localized in cytoplasmic foci in a pattern resembling P-body organization. Given that amoebic foci appear to be smaller and less rounded than those described in higher eukaryotes, we have named them "P-body-like structures". These foci contain additional mRNA degradation factors, including the EhCAF1 deadenylase and the EhAGO2-2 protein involved in RNA interference. Biochemical analysis revealed that EhCAF1 co-immunoprecipitated with EhXRN2 but not with EhDCP2 or EhAGO2-2, thus linking deadenylation to 5'-to-3' mRNA decay. The number of EhCAF1-containing foci significantly decreased after inhibition of transcription and translation with actinomycin D and cycloheximide, respectively. Furthermore, results of RNA-FISH assays showed that (i EhCAF1 colocalized with poly(A(+ RNA and (ii during silencing of the Ehpc4 gene by RNA interference, EhAGO2-2 colocalized with small interfering RNAs in cytoplasmic foci. Our observation of decapping, deadenylation and RNA interference proteins within P-body-like foci suggests that these structures have been conserved after originating in the early evolution of eukaryotic lineages. To the best of our knowledge, this is the first study to report on the localization of mRNA decay proteins within P

  18. Effects of cold-water immersion on VEGF mRNA and protein expression in heart and skeletal muscles of rats.

    Science.gov (United States)

    Kim, J C; Yi, H K; Hwang, P H; Yoon, J S; Kim, H J; Kawano, F; Ohira, Y; Kim, C K

    2005-04-01

    The effects of cold exposure on gene and protein expression of vascular endothelial growth factor (VEGF), in heart and skeletal muscles, were studied in male adult Wistar rats. Cold immersion was accomplished by submerging the rats in shoulder-deep water maintained at approximately 18 degrees C, either acutely (1 h) or chronically (1 h day(-1), 5 days week(-1) for 20 weeks). The expressions of VEGF mRNA and protein in heart, gastrocnemius, and soleus muscles were examined by Northern and Western blotting and competitive-polymerase chain reaction techniques. The expressions of VEGF mRNA and protein were markedly increased in cardiac muscle of the cold-immersed group, particularly in the 1-hour exposure group, whereas VEGF mRNA and protein in gastrocnemius were decreased significantly after an acute exposure. Although the protein level in gastrocnemius remained low in the chronically exposed group, the expression of mRNA of VEGF(165) with chronic exposure in this group returned to the control level and that of VEGF(206) was 15% greater than that in controls. The expression of mRNA for VEGF(165) in soleus was also lowered by acute cold exposure, although that for VEGF(206) was stable. However, VEGF protein was increased by 50%. After 20 weeks, all of these parameters were increased over the levels found in the controls. These results suggest that the VEGF gene may be a major regulatory factor in cardiac and skeletal muscle adaptation to the cold environment stimulating angiogenesis and thermogenesis.

  19. Molecular recognition of mRNA 5' cap by 3' poly(A)-specific ribonuclease (PARN) differs from interactions known for other cap-binding proteins.

    Science.gov (United States)

    Niedzwiecka, Anna; Nilsson, Per; Worch, Remigiusz; Stepinski, Janusz; Darzynkiewicz, Edward; Virtanen, Anders

    2016-04-01

    The mRNA 5' cap structure plays a pivotal role in coordination of eukaryotic translation and mRNA degradation. Poly(A)-specific ribonuclease (PARN) is a dimeric exoribonuclease that efficiently degrades mRNA 3' poly(A) tails while also simultaneously interacting with the mRNA 5' cap. The cap binding amplifies the processivity of PARN action. We used surface plasmon resonance kinetic analysis, quantitative equilibrium fluorescence titrations and circular dichroism to study the cap binding properties of PARN. The molecular mechanism of 5' cap recognition by PARN has been demonstrated to differ from interactions seen for other known cap-binding proteins in that: i) the auxiliary biological function of 5' cap binding by the 3' degrading enzyme is accomplished by negative cooperativity of PARN dimer subunits; ii) non-coulombic interactions are major factors in the complex formation; and iii) PARN has versatile activity toward alternative forms of the cap. These characteristics contribute to stabilization of the PARN-cap complex needed for the deadenylation processivity. Our studies provide a consistent biophysical basis for elucidation of the processive mechanism of PARN-mediated 3' mRNA deadenylation and provide a new framework to interpret the role of the 5' cap in mRNA degradation. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Rrp6p controls mRNA poly(a) tail length and its decoration with poly(a) binding proteins

    DEFF Research Database (Denmark)

    Schmid, Manfred; Poulsen, Mathias Bach; Olszewski, Pawel;

    2012-01-01

    Poly(A) (pA) tail binding proteins (PABPs) control mRNA polyadenylation, stability, and translation. In a purified system, S. cerevisiae PABPs, Pab1p and Nab2p, are individually sufficient to provide normal pA tail length. However, it is unknown how this occurs in more complex environments. Here ...

  1. T cell subsets and cytokines in allergic and non-allergic children. I. Analysis of IL-4, IFN-? and IL-13 mRNA expression and protein production

    NARCIS (Netherlands)

    Koning, H.; Neijens, H.J.; Baert, M.R.M.; Oranje, A.P.; Savelkoul, H.F.J.

    1997-01-01

    Interleukin 4 (IL-4) and IL-13 are key cytokines inducing switching to immunoglobulin E (IgE), whereas interferon (IFN-) acts inhibitory on this process. We analysed whether differences existed in IL-4, IFN- and IL-13 mRNA expression and protein production between T cells of children with allergic a

  2. Ekspresi level gen mRNA protein ekstraseluler otak embrio mencit black-6 uk-12 akibat induksi 2-methoxyethanol : analisis secara real time RT-PCR

    Directory of Open Access Journals (Sweden)

    Yulia Irnidayanti

    2012-02-01

    Full Text Available The aim of this research was to investigate impact of 2-methoxyethanol, a major industrial chemical, and its individual metaboliteson the expression DNA of the embryonic brain development of black-6 mice. The expression levels mRNA protein of GAPDH, Fibronectin,tenascin, vimentin, Neurofilamen, NCam between brain embrio treatment with 2-ME at gestation day 12 and Embryo control wereachieved. The Electroforesis DNA on brain Embryonic day 12 showed that there were expression of GAPDH (447bp, Fibronectin(462bp, NCAM (293 bp, Tenascin (416bp, Vimentin (327, Neurofilamen high (301bp, Neurofilamen medium (289bp, Neurofilamenlow (398bp. This Data not showed. The expression of level of mRNA for protein Vimentin at embryonic brain treatment at GD-12 is487 copies, meanwhile on the embryinoc brain control is 209 copies. This expression is tendency very higher than control. Anotherlevel of mRNA for protein fibronectin, NCAM, Tenascin, Neurofilament were tendency not differe between embryinoc brain treatmentsand control. Intermediate filaments, vimentin, is found in specific cell types in the developing and adult central nervous systems (CNS,particularly astrocytes. Recently, found that vimentin immunoreactivities were increased in astrocytes and/or macrophages in the spinalcords of rats with autoimmune inflammation. So that The higher level mRNA for protein vimentin caused by effect 2-methoxyethanol.Vimentin contribute to the repair of brain through the migration of activated cells and increased level vimentin at embryionic braintreatment with 2-ME.

  3. Green tea polyphenols improve cardiac muscle mRNA, and protein levels of signal pathways related to insulin and lipid metabolism and inflammation in insulin-resistant rats

    Science.gov (United States)

    Epidemiologic studies indicate that the consumption of green tea polyphenols (GTP) may reduce the risk of coronary artery disease. To explore the underlying mechanisms of action at the molecular level, we examined the effects of GTP on cardiac mRNA and protein levels of genes involved in insulin an...

  4. T cell subsets and cytokines in allergic and non-allergic children. I. Analysis of IL-4, IFN-? and IL-13 mRNA expression and protein production

    NARCIS (Netherlands)

    Koning, H.; Neijens, H.J.; Baert, M.R.M.; Oranje, A.P.; Savelkoul, H.F.J.

    1997-01-01

    Interleukin 4 (IL-4) and IL-13 are key cytokines inducing switching to immunoglobulin E (IgE), whereas interferon (IFN-) acts inhibitory on this process. We analysed whether differences existed in IL-4, IFN- and IL-13 mRNA expression and protein production between T cells of children with allergic

  5. Chronic corticosterone decreases brain-derived neurotrophic factor (BDNF) mRNA and protein in the hippocampus, but not in the frontal cortex, of the rat.

    Science.gov (United States)

    Jacobsen, Jacob P R; Mørk, Arne

    2006-09-19

    This study examined the effects of chronic corticosterone (32 mg/kg/day, s.c., 21 days) on brain-derived neurotrophic factor (BDNF) mRNA and protein in the frontal cortex and hippocampus of the rat. Because evidence suggests that BDNF is an important determinant of the function of the 5-hydroxytryptamine (5-HT) system, we also quantified tissue levels of 5-HT and its major metabolite, 5-hydroxyindoleacetic acid (5-HIAA), to investigate if changes in BDNF mRNA and protein paralleled changes in the 5-HT system. Corticosterone modestly decreased BDNF protein (-16.6%) in whole hippocampus and BDNF mRNA (-19%) in the CA3 area. In contrast, BDNF mRNA and protein in the frontal cortex were unchanged. In both the frontal cortex and hippocampus, tissue levels of 5-HT and 5-HIAA were increased and decreased, respectively. Combined, these data suggests that the effects of corticosterone on the BDNF system are not linked to the effects on the 5-HT systems. However, our findings do suggest that chronic corticosterone impairs hippocampal BDNF function, a finding with potential relevance for the hippocampal atrophy reported in major depression. Additionally, as inferred from the alterations in tissue levels of 5-HT and 5-HIAA, chronic corticosterone may influence the function of the 5-HT system.

  6. Excavation of a buried treasure--DNA, mRNA, miRNA and protein analysis in formalin fixed, paraffin embedded tissues.

    Science.gov (United States)

    Klopfleisch, R; Weiss, A T A; Gruber, A D

    2011-06-01

    Fresh or frozen tissue samples will always be the best tissue source for the analysis of nucleic acids and proteins from tissues. However, their long-term storage is expensive and laborious. Much interest has therefore been focused on the question whether the almost infinite resources of formalin fixed and paraffin embedded tissue samples in the archives of pathology and histology departments can be used for research on biomarkers and molecular mechanisms of disease. In recent years the methods and protocols for the extraction of DNA, mRNA, miRNA and proteins from formalin-fixed and paraffin-embedded tissue samples have improved enormously. Especially, the possibilities of analysing DNA and miRNA in FFPE have reached a level that allows their application as a first line approach in the search for biomarkers. In contrast, many questions remain in terms of quantification of mRNA and protein expression levels in formalin-fixed and paraffin-embedded tissue samples. This review gives an overview on current potentials and limitations of the quantification of DNA, miRNA, mRNA and the proteome in FFPE tissue samples. The chemical events during formalin fixation and paraffin embedding and alternatives to formalin fixation are described. In addition, methods and general problems of DNA, miRNA, mRNA and protein extraction and the current knowledge on the feasibility and accuracy of quantitative gene expression analysis in FFPE tissues is summarized.

  7. Disruption of the regulatory beta subunit of protein kinase CK2 in mice leads to a cell-autonomous defect and early embryonic lethality

    DEFF Research Database (Denmark)

    Buchou, Thierry; Vernet, Muriel; Blond, Olivier

    2003-01-01

    Protein kinase CK2 is a ubiquitous protein kinase implicated in proliferation and cell survival. Its regulatory beta subunit, CK2beta, which is encoded by a single gene in mammals, has been suspected of regulating other protein kinases. In this work, we show that knockout of the CK2beta gene in m....... Thus, our study demonstrates that in mammals, CK2beta is essential for viability at the cellular level, possibly because it acquired new functions during evolution.......Protein kinase CK2 is a ubiquitous protein kinase implicated in proliferation and cell survival. Its regulatory beta subunit, CK2beta, which is encoded by a single gene in mammals, has been suspected of regulating other protein kinases. In this work, we show that knockout of the CK2beta gene...

  8. Murrayafoline A attenuates the Wnt/{beta}-catenin pathway by promoting the degradation of intracellular {beta}-catenin proteins

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Hyuk; Gwak, Jungsug; Cho, Munju; Ryu, Min-Jung [PharmacoGenomics Research Center, Inje University, Busan 614-735 (Korea, Republic of); Lee, Jee-Hyun; Kim, Sang Kyum; Kim, Young Ho [College of Pharmacy, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Lee, Gye Won [Department of Pharmaceutical Engineering, Konyang University, Nonsan 320-711 (Korea, Republic of); Yun, Mi-Young [Department of Beauty Health Care, Daejeon University, Daejeon 305-764 (Korea, Republic of); Cuong, Nguyen Manh [Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology, Hanoi (Viet Nam); Shin, Jae-Gook [PharmacoGenomics Research Center, Inje University, Busan 614-735 (Korea, Republic of); Song, Gyu-Yong, E-mail: gysong@cnu.ac.kr [College of Pharmacy, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Oh, Sangtaek, E-mail: ohsa@inje.ac.kr [PharmacoGenomics Research Center, Inje University, Busan 614-735 (Korea, Republic of)

    2010-01-01

    Molecular lesions in Wnt/{beta}-catenin signaling and subsequent up-regulation of {beta}-catenin response transcription (CRT) occur frequently during the development of colon cancer. To identify small molecules that suppress CRT, we screened natural compounds in a cell-based assay for detection of TOPFalsh reporter activity. Murrayafoline A, a carbazole alkaloid isolated from Glycosmis stenocarpa, antagonized CRT that was stimulated by Wnt3a-conditioned medium (Wnt3a-CM) or LiCl, an inhibitor of glycogen synthase kinase-3{beta} (GSK-3{beta}), and promoted the degradation of intracellular {beta}-catenin without altering its N-terminal phosphorylation at the Ser33/37 residues, marking it for proteasomal degradation, or the expression of Siah-1, an E3 ubiquitin ligase. Murrayafoline A repressed the expression of cyclin D1 and c-myc, which is known {beta}-catenin/T cell factor (TCF)-dependent genes and thus inhibited the proliferation of various colon cancer cells. These findings indicate that murrayafoline A may be a potential chemotherapeutic agent for use in the treatment of colon cancer.

  9. Human DDX3 interacts with the HIV-1 Tat protein to facilitate viral mRNA translation.

    Directory of Open Access Journals (Sweden)

    Ming-Chih Lai

    Full Text Available Nuclear export and translation of intron-containing viral mRNAs are required for HIV-1 gene expression and replication. In this report, we provide evidence to show that DDX3 regulates the translation of HIV-1 mRNAs. We found that knockdown of DDX3 expression effectively inhibited HIV-1 production. Translation of HIV-1 early regulatory proteins, Tat and rev, was impaired in DDX3-depleted cells. All HIV-1 transcripts share a highly structured 5' untranslated region (UTR with inhibitory elements on translation of viral mRNAs, yet DDX3 promoted translation of reporter mRNAs containing the HIV-1 5' UTR, especially with the transactivation response (TAR hairpin. Interestingly, DDX3 directly interacts with HIV-1 Tat, a well-characterized transcriptional activator bound to the TAR hairpin. HIV-1 Tat is partially targeted to cytoplasmic stress granules upon DDX3 overexpression or cell stress conditions, suggesting a potential role of Tat/DDX3 complex in translation. We further demonstrated that HIV-1 Tat remains associated with translating mRNAs and facilitates translation of mRNAs containing the HIV-1 5' UTR. Taken together, these findings indicate that DDX3 is recruited to the TAR hairpin by interaction with viral Tat to facilitate HIV-1 mRNA translation.

  10. Human DDX3 interacts with the HIV-1 Tat protein to facilitate viral mRNA translation.

    Science.gov (United States)

    Lai, Ming-Chih; Wang, Shainn-Wei; Cheng, Lie; Tarn, Woan-Yuh; Tsai, Shaw-Jenq; Sun, H Sunny

    2013-01-01

    Nuclear export and translation of intron-containing viral mRNAs are required for HIV-1 gene expression and replication. In this report, we provide evidence to show that DDX3 regulates the translation of HIV-1 mRNAs. We found that knockdown of DDX3 expression effectively inhibited HIV-1 production. Translation of HIV-1 early regulatory proteins, Tat and rev, was impaired in DDX3-depleted cells. All HIV-1 transcripts share a highly structured 5' untranslated region (UTR) with inhibitory elements on translation of viral mRNAs, yet DDX3 promoted translation of reporter mRNAs containing the HIV-1 5' UTR, especially with the transactivation response (TAR) hairpin. Interestingly, DDX3 directly interacts with HIV-1 Tat, a well-characterized transcriptional activator bound to the TAR hairpin. HIV-1 Tat is partially targeted to cytoplasmic stress granules upon DDX3 overexpression or cell stress conditions, suggesting a potential role of Tat/DDX3 complex in translation. We further demonstrated that HIV-1 Tat remains associated with translating mRNAs and facilitates translation of mRNAs containing the HIV-1 5' UTR. Taken together, these findings indicate that DDX3 is recruited to the TAR hairpin by interaction with viral Tat to facilitate HIV-1 mRNA translation.

  11. Daxx-like protein of Drosophila interacts with Dmp53 and affects longevity and Ark mRNA level.

    Science.gov (United States)

    Bodai, László; Pardi, Norbert; Ujfaludi, Zsuzsanna; Bereczki, Orsolya; Komonyi, Orbán; Balint, Eva; Boros, Imre M

    2007-12-14

    Daxx-like protein (DLP), the Drosophila homolog of Daxx, binds Drosophila melanogaster p53 (Dmp53) through its C-terminal region. We generated DLP mutants and found that although DLP expression is developmentally regulated, it is not essential for the execution of the developmental program. The effects DLP mutations show in the loss of heterozygosity assay and on phenotypes resulting from Dmp53 overexpression indicate a genetic interaction between DLP and Dmp53. In contrast to Dmp53 mutants, however, loss of DLP does not result in radiosensitivity indicating that it does not play an essential role in the activation of Dmp53-dependent response after ionizing radiation, and DLP is also not required for the irradiation-induced activation of reaper. In contrast, DLP is involved in the transcriptional regulation of Ark, because Ark mRNA level is decreased in DLP mutants and increased upon ectopic overexpression of DLP. Interestingly, DLP mutants have reduced longevity and reduced female fertility. Altogether, our data suggest complex functions for DLP, which include an anti-apoptotic effect exerted through repression of some Dmp53 functions, and activation of some proapoptotic genes.

  12. Identification of TEL-AML1 (ETV6-RUNX1 associated DNA and its impact on mRNA and protein output using ChIP, mRNA expression arrays and SILAC

    Directory of Open Access Journals (Sweden)

    Yvonne Linka

    2014-12-01

    Full Text Available The contribution of the most common reciprocal translocation in childhood B-cell precursor leukemia t(12;21(p13;q22 to leukemia development is still under debate. Direct as well as secondary indirect effects of the TEL-AML1 fusion protein are commonly recorded by using cell lines and patient samples, often bearing the TEL-AML1 fusion protein for decades. To identify direct targets of the fusion protein a short-term induction of TEL-AML1 is needed. We here describe in detail the experimental procedure, quality controls and contents of the ChIP, mRNA expression and SILAC datasets associated with the study published by Linka and colleagues in the Blood Cancer Journal [1] utilizing a short term induction of TEL-AML1 in an inducible precursor B-cell line model.

  13. Marked effect of beta-lactoglobulin polymorphism on the ratio of casein to total protein in milk.

    Science.gov (United States)

    Lundén, A; Nilsson, M; Janson, L

    1997-11-01

    The relationship between genetic variants for milk protein and the composition of milk was analyzed on 4475 repeated milk samples from individual cows; 371 dairy cows of the Swedish Red and White breed and 204 cows of the Swedish Holstein breed were used. The registrations included percentages of casein, protein, fat, and lactose in combination with milk yield and SCC. The genotype of individual cows for alpha(s1)-CN, beta-CN, kappa-CN, and beta-LG was determined by alkaline and acidic PAGE. A mixed animal model was used for the analysis; beta-LG and aggregate casein genotypes were included simultaneously as separate fixed effects in the statistical model. The results suggest a positive additive effect of the beta-LG B allele on casein content and on the ratio of casein to total protein. For the latter trait, the beta-LG genotype accounted for a relatively large part of the phenotypic variance, corresponding to a reduction in residual variance of 11% when included in the model. The corresponding value for casein content was 0.5%. The lack of unfavorable associations between milk protein variants and the traits included in this study makes the beta-LG gene an obvious candidate when the breeding objective is improved conversion of milk protein into cheese.

  14. Porcine reproductive and respiratory syndrome virus nonstructural protein 1beta modulates host innate immune response by antagonizing IRF3 activation.

    Science.gov (United States)

    Beura, Lalit K; Sarkar, Saumendra N; Kwon, Byungjoon; Subramaniam, Sakthivel; Jones, Clinton; Pattnaik, Asit K; Osorio, Fernando A

    2010-02-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) infection of swine leads to a serious disease characterized by a delayed and defective adaptive immune response. It is hypothesized that a suboptimal innate immune response is responsible for the disease pathogenesis. In the study presented here we tested this hypothesis and identified several nonstructural proteins (NSPs) with innate immune evasion properties encoded by the PRRS viral genome. Four of the total ten PRRSV NSPs tested were found to have strong to moderate inhibitory effects on beta interferon (IFN-beta) promoter activation. The strongest inhibitory effect was exhibited by NSP1 followed by, NSP2, NSP11, and NSP4. We focused on NSP1alpha and NSP1beta (self-cleavage products of NSP1 during virus infection) and NSP11, three NSPs with strong inhibitory activity. All of three proteins, when expressed stably in cell lines, strongly inhibited double-stranded RNA (dsRNA) signaling pathways. NSP1beta was found to inhibit both IFN regulatory factor 3 (IRF3)- and NF-kappaB-dependent gene induction by dsRNA and Sendai virus. Mechanistically, the dsRNA-induced phosphorylation and nuclear translocation of IRF3 were strongly inhibited by NSP1beta. Moreover, when tested in a porcine myelomonocytic cell line, NSP1beta inhibited Sendai virus-mediated activation of porcine IFN-beta promoter activity. We propose that this NSP1beta-mediated subversion of the host innate immune response plays an important role in PRRSV pathogenesis.

  15. Estrogen protects neuronal cells from amyloid beta-induced apoptosis via regulation of mitochondrial proteins and function

    Directory of Open Access Journals (Sweden)

    Iwamoto Sean

    2006-11-01

    Full Text Available Abstract Background Neurodegeneration in Alzheimer's disease is associated with increased apoptosis and parallels increased levels of amyloid beta, which can induce neuronal apoptosis. Estrogen exposure prior to neurotoxic insult of hippocampal neurons promotes neuronal defence and survival against neurodegenerative insults including amyloid beta. Although all underlying molecular mechanisms of amyloid beta neurotoxicity remain undetermined, mitochondrial dysfunction, including altered calcium homeostasis and Bcl-2 expression, are involved in neurodegenerative vulnerability. Results In this study, we investigated the mechanism of 17β-estradiol-induced prevention of amyloid beta-induced apoptosis of rat hippocampal neuronal cultures. Estradiol treatment prior to amyloid beta exposure significantly reduced the number of apoptotic neurons and the associated rise in resting intracellular calcium levels. Amyloid beta exposure provoked down regulation of a key antiapoptotic protein, Bcl-2, and resulted in mitochondrial translocation of Bax, a protein known to promote cell death, and subsequent release of cytochrome c. E2 pretreatment inhibited the amyloid beta-induced decrease in Bcl-2 expression, translocation of Bax to the mitochondria and subsequent release of cytochrome c. Further implicating the mitochondria as a target of estradiol action, in vivo estradiol treatment enhanced the respiratory function of whole brain mitochondria. In addition, estradiol pretreatment protected isolated mitochondria against calcium-induced loss of respiratory function. Conclusion Therefore, we propose that estradiol pretreatment protects against amyloid beta neurotoxicity by limiting mitochondrial dysfunction via activation of antiapoptotic mechanisms.

  16. G-Protein Inwardly Rectifying Potassium Channel 1 (GIRK1 Knockdown Decreases Beta-Adrenergic, MAP Kinase and Akt Signaling in the MDA-MB-453 Breast Cancer Cell Line

    Directory of Open Access Journals (Sweden)

    Michael W. Hance

    2008-01-01

    Full Text Available Previous data from our laboratory have indicated that there is a functional link between the beta-adrenergic receptor signaling pathway and the G-protein inwardly rectifying potassium channel (GIRK1 in breast cancer cell lines and that these pathways are involved in growth regulation of these cells. To determine functionality, MDA-MB-453 breast cancer cells were stimulated with ethanol, known to open GIRK channels. Decreased GIRK1 protein levels were seen after treatment with 0.12% ethanol. In addition, serum-free media completely inhibited GIRK1 protein expression. This data indicates that there are functional GIRK channels in breast cancer cells and that these channels are involved in cellular signaling. In the present research, to further define the signaling pathways involved, we performed RNA interference (siRNA studies. Three stealth siRNA constructs were made starting at bases 1104, 1315, and 1490 of the GIRK1 sequence. These constructs were transfected into MDA-MB-453 cells, and both RNA and protein were isolated. GIRK1, β2-adrenergic and 18S control levels were determined using real-time PCR 24 hours after transfection. All three constructs decreased GIRK1 mRNA levels. However, β2 mRNA levels were unchanged by the GIRK1 knockdown. GIRK1 protein levels were also reduced by the knockdown, and this knockdown led to decreases in beta-adrenergic, MAP kinase and Akt signaling.

  17. Correlative mRNA and protein expression of middle and inner ear inflammatory cytokines during mouse acute otitis media.

    Science.gov (United States)

    Trune, Dennis R; Kempton, Beth; Hausman, Frances A; Larrain, Barbara E; MacArthur, Carol J

    2015-08-01

    Although the inner ear has long been reported to be susceptible to middle ear disease, little is known of the inflammatory mechanisms that might cause permanent sensorineural hearing loss. Recent studies have shown inner ear tissues are capable of expressing inflammatory cytokines during otitis media. However, little quantitative information is available concerning cytokine gene expression in the inner ear and the protein products that result. Therefore, this study was conducted of mouse middle and inner ear during acute otitis media to measure the relationship between inflammatory cytokine genes and their protein products with quantitative RT-PCR and ELISA, respectively. Balb/c mice were inoculated transtympanically with heat-killed Haemophilus influenzae and middle and inner ear tissues collected for either quantitative RT-PCR microarrays or ELISA multiplex arrays. mRNA for several cytokine genes was significantly increased in both the middle and inner ear at 6 h. In the inner ear, these included MIP-2 (448 fold), IL-6 (126 fold), IL-1β (7.8 fold), IL-10 (10.7 fold), TNFα (1.8 fold), and IL-1α (1.5 fold). The 24 h samples showed a similar pattern of gene expression, although generally at lower levels. In parallel, the ELISA showed the related cytokines were present in the inner ear at concentrations higher by 2-122 fold higher at 18 h, declining slightly from there at 24 h. Immunohistochemistry with antibodies to a number of these cytokines demonstrated they occurred in greater amounts in the inner ear tissues. These findings demonstrate considerable inflammatory gene expression and gene products in the inner ear following acute otitis media. These higher cytokine levels suggest one potential mechanism for the permanent hearing loss seen in some cases of acute and chronic otitis media.

  18. Effects of breeds and dietary protein levels on the growth performance, energy expenditure and expression of avUCP mRNA in chickens.

    Science.gov (United States)

    Li, Qihua; Xu, Zhiqiang; Liu, L; Yu, Hongxin; Rong, Hua; Tao, Linli; Zhang, Xi; Chen, Xiaobo; Gu, Dahai; Fan, Yueyuan; Li, Xiaoqin; Ge, Changrong; Tian, Yunbo; Jia, Junjing

    2013-04-01

    The physiological mechanisms of thermogenesis, energy balance and energy expenditure are poorly understood in poultry. The aim of this study was designed to investigate the physiological roles of avian uncoupling protein (avUCP) regulating in energy balance and thermogenesis by using three chicken breeds of existence striking genetic difference and feeding with different dietary protein levels. Three chicken breeds including broilers, hybrid chickens, and non-selection Wuding chickens were used in this study. Total 150 chicks of 1 day of age, with 50 from each breed were reared under standard conditions on starter diets to 30 days. At 30 days of age, forty chicks from each breed chicks were divided into two groups. One group was fed low protein diet (LP, 17.0 %), and the other group was fed high protein diet (HP, 19.5 %) for 60 days. Wuding chickens showed the lowest feed conversion efficiency (FCE) and the highest expressions of avUCP mRNA association with high plasma T3 and insulin concentrations. Hybrid chickens showed the lowest expressions of avUCP mRNA association with high FCE and energy efficiency. Expressions of avUCP mRNA association with diet-induced thermogenesis (DIT) were only observed in broiler and hybrid chickens. The expressions of avUCP mRNA were positive association with plasma insulin, T3 and NEFA concentrations. Age influence on the expression of avUCP mRNA were observed only for hybrid and broiler chickens. It seems that both roles of avUCP regulation thermogenesis and lipid utilisation as fuel were observed in the present study response to variation in dietary protein and breeds.

  19. Photobiomodulation on Bax and Bcl-2 Proteins and SIRT1/PGC-1α Axis mRNA Expression Levels of Aging Rat Skeletal Muscle

    Directory of Open Access Journals (Sweden)

    Fang-Hui Li

    2014-01-01

    Full Text Available Objective. This study aimed to analyze the effects of low level laser irradiation (LLLI on Bax and IGF-1 and Bcl-2 protein contents and SIRT1/PGC-1α axis mRNA expression levels to prevent sarcopenia in aged rats. Material and Methods. Twenty female Sprague Dawley rats (18 months old were randomly divided into two groups (n=10 per group: control (CON and LLLI groups. The gallium-aluminum-arsenium (GaAlAs laser irradiation at 810 nm was used in the single point contact mode (3.75 J/cm2; 0.4 cm2; 125 mW/cm2; 30 s. Bax, Bcl-2, and IGF-1 proteins and SIRT1/PGC-1α axis mRNA expression were assessed 24 h after LLLI on gastrocnemius in aged rat. Results. Gastrocnemius muscle weights, gastrocnemius mass/body mass, Bcl-2/BAX ratio, Bcl-2 protein, IGF-1 protein, and the mRNA contents in SIRT1, PGC-1α, NRF1, TMF, and SOD2 were significantly (P<0.05 increased by LLLI compared to CON group without LLLI. However, levels of BAX protein and caspase 3 mRNA were significantly attenuated by LLLI compared to CON group (P<0.05. Conclusion. LLLI at 810 nm inhibits sarcopenia associated with upregulation of Bcl-2/BAX ratio and IGF-1 and SIRT1/PGC-1α axis mRNA expression in aged rats. This indicates that LLLI has potential to decrease progression of myocyte apoptosis in sarcopenic muscles.

  20. Differential distribution of G-protein beta-subunits in brain: an immunocytochemical analysis.

    Science.gov (United States)

    Brunk, I; Pahner, I; Maier, U; Jenner, B; Veh, R W; Nürnberg, B; Ahnert-Hilger, G

    1999-05-01

    Heterotrimeric G proteins play central roles in signal transduction of neurons and other cells. The variety of their alpha-, beta-, and gamma-subunits allows numerous combinations thereby confering specificity to receptor-G-protein-effector interactions. Using antisera against individual G-protein beta-subunits we here present a regional and subcellular distribution of Gbeta1, Gbeta2, and Gbeta5 in rat brain. Immunocytochemical specificity of the subtype-specific antisera is revealed in Sf9 cells infected with various G-protein beta-subunits. Since Gbeta-subunits together with a G-protein gamma-subunit affect signal cascades we include a distribution of the neuron-specific Ggamma2- and Ggamma3-subunits in selected brain areas. Gbeta1, Gbeta2, and Gbeta5 are preferentially distributed in the neuropil of hippocampus, cerebellum and spinal cord. Gbeta2 is highly concentrated in the mossy fibres of dentate gyrus neurons ending in the stratum lucidum of hippocampal CA3-area. High amounts of Gbeta2 also occur in interneurons innervating spinal cord alpha-motoneurons. Gbeta5 is differentially distributed in all brain areas studied. It is found in the pyramidal cells of hippocampal CA1-CA3 as well as in the granule cell layer of dentate gyrus and in some interneurons. In the spinal cord Gbeta5 in contrast to Gbeta2 concentrates around alpha-motoneurons. In cultivated mouse hippocampal and hypothalamic neurons Gbeta2 and Gbeta5 are found in different subcellular compartments. Whereas Gbeta5 is restricted to the perikarya, Gbeta2 is also found in processes and synaptic contacts where it partially colocalizes with the synaptic vesicle protein synaptobrevin. An antiserum recognizing Ggamma2 and Ggamma3 reveals that these subunits are less expressed in hippocampus and cerebellum. Presumably this antiserum specifically recognizes Ggamma2 and Ggamma3 in combinations with certain G alphas and/or Gbetas. The widespread but regionally and cellularly rather different distribution of

  1. Polypyrimidine tract-binding protein is critical for the turnover and subcellular distribution of CD40 ligand mRNA in CD4+ T cells.

    Science.gov (United States)

    Matus-Nicodemos, Rodrigo; Vavassori, Stefano; Castro-Faix, Moraima; Valentin-Acevedo, Anibal; Singh, Karnail; Marcelli, Valentina; Covey, Lori R

    2011-02-15

    CD40L (CD154) is regulated at the posttranscriptional level by an activation-induced process that results in a highly stable transcript at extended times of T cell activation. Transcript stability is mediated by polypyrimidine tract-binding protein (PTB)-containing complexes (complex I and II) that bind to three adjacent CU-rich sequences within the 3' untranslated region. To assess the role of PTB in the expression and distribution of CD40L mRNA, PTB was targeted using short hairpin RNA in both primary T cells and a T cell line that recapitulates the stability phase of regulated CD40L mRNA decay. PTB knockdown resulted in a marked decrease in the mRNA stability that resulted in lowered CD40L surface expression. PTB was also critical for appropriate distribution of CD40L mRNA between the nucleus and cytoplasm and in the cytoplasm between the cytosol and the translating polysomes. The activation-induced formation of PTB-specific ribonucleoprotein complexes was observed only with cytoplasmic and not nuclear PTB indicating functional differences in the protein defined by cellular localization. Finally, we observed that cytoplasmic and nuclear PTB isoforms were differentially modified relative to each other and that the changes in cytoplasmic PTB were consistent with activation-induced phosphorylation. Together this work suggests that differentially modified PTB regulates CD40L expression at multiple steps by 1) retaining CD40L mRNA in the nucleus, 2) directly regulating mRNA stability at late times of activation, and 3) forming a ribonuclear complex that preferentially associates with translating ribosomes thus leading to an enhanced level of CD40L protein.

  2. mRNA levels of BACE1 and its interacting proteins, RTN3 and PPIL2, correlate in human post mortem brain tissue.

    Science.gov (United States)

    Beyer, N; Coulson, D T R; Quinn, J G; Brockbank, S; Hellemans, J; Irvine, G B; Ravid, R; Johnston, J A

    2014-08-22

    β-Site amyloid precursor protein cleaving enzyme (BACE1) is the rate-limiting enzyme for production of Aβ peptides, proposed to drive the pathological changes found in Alzheimer's disease (AD). Reticulon 3 (RTN3) is a negative modulator of BACE1 (β-secretase) proteolytic activity, while peptidylprolyl isomerase (cyclophilin)-like 2 (PPIL2) positively regulated BACE1 gene expression in a cell-based assay. This study aimed to analyze RTN3 and PPIL2 mRNA levels in four brain regions from individuals with AD and controls. BACE1 mRNA had been previously quantified in the samples, as had glial fibrillary acidic protein (GFAP) and neuron-specific enolase (NSE), to track changing cell populations in the tissue. mRNA levels in the human post mortem brain tissue were assayed using quantitative real-time polymerase chain reaction (qPCR) and qbase(PLUS), employing validated stably expressed reference genes. No differences in RTN3 or PPIL2 mRNA levels were found in individuals with AD, compared to controls. Both RTN3 and PPIL2 mRNA levels correlated significantly with BACE1 mRNA and all three showed similar disease stage-dependent changes with respect to NSE and GFAP. These findings indicated that the in vitro data demonstrating an effect of PPIL2 on BACE1 expression have functional relevance in vivo. Further research into BACE1-interacting proteins could provide a fruitful approach to the modulation of this protease and consequently Aβ production.

  3. Tumors from rats given 1,2-dimethylhydrazine plus chlorophyllin or indole-3-carbinol contain transcriptional changes in beta-catenin that are independent of beta-catenin mutation status.

    Science.gov (United States)

    Wang, Rong; Dashwood, W Mohaiza; Bailey, George S; Williams, David E; Dashwood, Roderick H

    2006-10-10

    Tumors induced in the rat by 1,2-dimethylhydrazine (DMH) contain mutations in beta-catenin, but the spectrum of such mutations can be influenced by phytochemicals such as chlorophyllin (CHL) and indole-3-carbinol (I3C). In the present study, we determined the mutation status of beta-catenin in more than 50 DMH-induced colon tumors and small intestine tumors, and compared this with the concomitant expression of beta-catenin mRNA using quantitative real-time RT-PCR analysis. In total, 19/57 (33%) of the tumors harbored mutations in beta-catenin, and 14/19 (74%) of the genetic changes substituted amino acids adjacent to Ser33, a key site for phosphorylation and beta-catenin degradation. These tumors were found to express a 10-fold range of beta-catenin mRNA levels, independent of the beta-catenin mutation status and phytochemical exposure, i.e. CHL or I3C given post-initiation. However, beta-catenin mRNA levels were strongly correlated with mRNA levels of c-myc, c-jun and cyclin D1, which are targets of beta-catenin/Tcf signaling. Tumors with the highest levels of beta-catenin mRNA often had over-expressed beta-catenin protein, and those with lower beta-catenin mRNA typically had low beta-catenin protein expression, but there were exceptions (high beta-catenin mRNA/low beta-catenin protein, or vice versa). We conclude that DMH-induced mutations stabilize beta-catenin protein in tumors, which increase c-myc, c-jun and cyclin D1, but there also can be over-expression of beta-catenin itself at the mRNA level, contributing to high beta-catenin protein levels. Similar findings have been reported in primary human colon cancers and their liver metastases, compared with matched normal-looking tissue. Thus, further studies are warranted on the mechanisms that upregulate beta-catenin at the transcriptional level in human and rodent colon cancers.

  4. Nitrosative stress mediated misfolded protein aggregation mitigated by Na-D-{beta}-hydroxybutyrate intervention

    Energy Technology Data Exchange (ETDEWEB)

    Kabiraj, Parijat; Pal, Rituraj [Department of Chemistry, University of Texas at El Paso, El Paso, TX (United States); Varela-Ramirez, Armando [Department of Biological Sciences, Border Biomedical Research Center, University of Texas at El Paso, El Paso, TX (United States); Miranda, Manuel [Department of Biological Sciences, University of Texas at El Paso, El Paso, TX (United States); Narayan, Mahesh, E-mail: mnarayan@utep.edu [Department of Chemistry, University of Texas at El Paso, El Paso, TX (United States)

    2012-09-28

    Highlights: Black-Right-Pointing-Pointer Rotenone is a model for inducing apoptosis and synphilin-1 accumulation in Parkinson Prime s studies. Black-Right-Pointing-Pointer The metabolite sodium betahydroxybutryate mitigates these effects in SHSY5Y cell lines. Black-Right-Pointing-Pointer Results reveal a novel and innate mechanism to prevent neurodegeneration/cell death. -- Abstract: Mitochondrial dysfunction, leading to elevated levels of reactive oxygen species, is associated with the pathogenesis of neurodegenerative disorders. Rotenone, a mitochondrial stressor induces caspase-9 and caspase-3 activation leading proteolytic cleavage of substrate nuclear poly(ADP-ribose) polymerase (PARP). PARP cleavage is directly related to apoptotic cell death. In this study, we have monitored the aggregation of green-fluorescent protein (GFP)-tagged synphilin-1, as a rotenone-induced Parkinsonia-onset biomarker. We report that the innate ketone body, Na-D-{beta}-hydroxybutyrate (Na{beta}HB) reduces markedly the incidence of synphilin-1 aggregation. Furthermore, our data reveal that the metabolic byproduct also prevents rotenone-induced caspase-activated apoptotic cell death in dopaminergic SH-SY5Y cells. Together, these results suggest that Na{beta}HB is neuroprotective; it attenuates effects originating from mitochondrial insult and can serve as a scaffold for the design and development of sporadic neuropathies.

  5. Oxidative stress induces macroautophagy of amyloid beta-protein and ensuing apoptosis

    DEFF Research Database (Denmark)

    Zheng, Lin; Kågedal, Katarina; Dehvari, Nodi;

    2009-01-01

    There is increasing evidence for the toxicity of intracellular amyloid beta-protein (Abeta) to neurons and the involvement of lysosomes in this process in Alzheimer disease (AD). We have recently shown that oxidative stress, a recognized determinant of AD, enhances macroautophagy and leads...... to intralysosomal accumulation of Abeta in cultured neuroblastoma cells. We hypothesized that oxidative stress promotes AD by stimulating macroautophagy of Abeta that further may induce cell death by destabilizing lysosomal membranes. To investigate such possibility, we compared the effects of hyperoxia (40...

  6. Conformational intermediate of the amyloidogenic protein beta 2-microglobulin at neutral pH

    DEFF Research Database (Denmark)

    Heegaard, N H; Sen, J W; Kaarsholm, N C

    2001-01-01

    electrophoresis that two conformers spontaneously exist in aqueous buffers at neutral pH. Upon treatment of wild-type beta(2)-microglobulin with acetonitrile or trifluoroethanol, two conformations were also observed. These conformations were in equilibrium dependent on the sample temperature and the percentage...... conformation with the dyes 8-anilino-1-naphthalene sulfonic acid and Congo red. The observations may relate to early folding events prior to amyloid fibrillation and facilitate the development of methods to detect and inhibit pro-amyloid protein and peptide conformations....

  7. Fatty acid biosynthesis in Pseudomonas aeruginosa: cloning and characterization of the fabAB operon encoding beta-hydroxyacyl-acyl carrier protein dehydratase (FabA) and beta-ketoacyl-acyl carrier protein synthase I (FabB).

    OpenAIRE

    Hoang, T.T.; Schweizer, H P

    1997-01-01

    The Pseudomonas aeruginosa fabA and fabB genes, encoding beta-hydroxyacyl-acyl carrier protein dehydratase and beta-ketoacyl-acyl carrier protein synthase I, respectively, were cloned, sequenced, and expressed in Escherichia coli. Northern analysis demonstrated that fabA and fabB are cotranscribed and most probably form a fabAB operon. The FabA and FabB proteins were similar in size and amino acid composition to their counterparts from Escherichia coli and to the putative homologs from Haemop...

  8. Water Sparing in Chronic Ethanol Exposure is Associated With Elevated Renal Estrogen Receptor Beta and Vasopressin V2 Receptor mRNA in the Female Rate

    Science.gov (United States)

    2007-12-01

    quality as a thesis for the degree of Master of Science in Medical and Molecular Physiology. THESIS COMMITTEE Chairperson ii TABLE OF CONTENTS List of...of biology = Revista brasleira de biologia 62, 609-614 20. Bevan, D. R. (1978) Osmometry. 1. Terminology and principles of measurement. Anaesthesia 33... molecular endocrinology 24, 145-155 32. Suzuki, S., and Handa, R. J. (2004) Regulation of estrogen receptor-beta expression in the female rat

  9. Inhibitory effect of amygdalin on lipopolysaccharide-inducible TNF-alpha and IL-1beta mRNA expression and carrageenan-induced rat arthritis.

    Science.gov (United States)

    Hwang, Hye-Jeong; Lee, Hye-Jung; Kim, Chang-Ju; Shim, Insop; Hahm, Dae-Hyun

    2008-10-01

    Amygdalin is a cyanogenic glycoside plant compound found in the seeds of rosaceous stone fruits. We evaluated the antiinflammatory and analgesic activities of amygdalin, using an in vitro lipopolysaccharide (LPS)-induced cell line and a rat model with carrageenan-induced ankle arthritis. One mM amygdalin significantly inhibited the expression of TNF-alpha and IL-1beta mRNAs in LPS-treated RAW 264.7 cells. Amygdalin (0.005, 0.05, and 0.1 mg/kg) was intramuscularly injected immediately after the induction of carrageenan-induced arthritic pain in rats, and the anti-arthritic effect of amygdalin was assessed by measuring the weight distribution ratio of the bearing forces of both feet and the ankle circumference, and by analyzing the expression levels of three molecular markers of pain and inflammation (c-Fos, TNF-alpha, and IL-1beta) in the spinal cord. The hyperalgesia of the arthritic ankle was alleviated most significantly by the injection of 0.005 mg/kg amygdalin. At this dosage, the expressions of c-Fos, TNF-alpha, and IL-1beta in the spinal cord were significantly inhibited. However, at dosage greater than 0.005 mg/kg, the painrelieving effect of amygdalin was not observed. Thus, amygdalin treatment effectively alleviated responses to LPStreatment in RAW 264.7 cells and carrageenan-induced arthritis in rats, and may serve as an analgesic for relieving inflammatory pain.

  10. Differential and time-dependent expression of monocyte chemoattractant protein-1 mRNA by astrocytes and macrophages in rat brain : Effects of ischemia and peripheral lipopolysaccharide administration

    NARCIS (Netherlands)

    Gourmala, NG; Buttini, M; Limonta, S; Sauter, A; Boddeke, HWGM

    1997-01-01

    Increasing evidence indicates a key role of chemoattractant cytokines in the accumulation of leukocytes in the central nervous system (CNS) during the course of inflammatory processes. Monocyte chemoattractant protein (MCP-1/JE), a member of the beta-chemokine (C-C chemokine) family, functions as a

  11. GPI-80, a beta2 integrin associated glycosylphosphatidylinositol-anchored protein, concentrates on pseudopodia without association with beta2 integrin during neutrophil migration.

    Science.gov (United States)

    Yoshitake, Hiroshi; Takeda, Yuji; Nitto, Takeaki; Sendo, Fujiro; Araki, Yoshihiko

    2003-01-01

    Previously, we identified a glycosylphosphatidylinositol (GPI)-anchored protein, designated GPI-80, present on human neutrophils and monocytes. GPI-80 is physically associated with beta2 integrin on the surface of human neutrophils and may be a regulator of neutrophil adherence and migration. However, it is not yet known how GPI-80 regulates cell adhesion and migration. To investigate the physiological role(s) of GPI-80, we examined the topological relationship of GPI-80 and the beta2 integrin subunit (CD18) on resting and migrating human neutrophils by confocal laser microscopy. On resting neutrophils, GPI-80 was evenly distributed on the cell surface and was associated with CD18. On the other hand, during the early phase of migration (5 - 30 minutes), GPI-80 was detected on cell bodies and also on pseudopodia, but CD18 was detected only on cell bodies, where it was associated with GPI-80. In the late phase of migration (60 minutes), GPI-80 was detected only on pseudopodia and its association with CD18 was hardly observed. Furthermore, some of the GPI-80 on pseudopodia of migrating neutrophils during the late phase was associated with urokinase-type plasminogen activator receptor (uPAR), a regulator of beta2 integrin-dependent adherence and migration. The distribution of GPI-80 on cell surfaces is similar to that of uPAR. These observations suggest that GPI-80 belongs to the beta2 integrin-associated GPI-anchored protein family, which has regulatory activity in cell adherence.

  12. Proteins with an alpha/beta hydrolase fold: Relationships between subfamilies in an ever-growing superfamily.

    Science.gov (United States)

    Lenfant, Nicolas; Hotelier, Thierry; Bourne, Yves; Marchot, Pascale; Chatonnet, Arnaud

    2013-03-25

    Alpha/beta hydrolases function as hydrolases, lyases, transferases, hormone precursors or transporters, chaperones or routers of other proteins. The amount of structural and functional available data related to this protein superfamily expands exponentially, as does the number of proteins classified as alpha/beta hydrolases despite poor sequence similarity and lack of experimental data. However the superfamily can be rationally divided according to sequence or structural homologies, leading to subfamilies of proteins with potentially similar functions. Since the discovery of proteins homologous to cholinesterases but devoid of enzymatic activity (e.g., the neuroligins), divergent functions have been ascribed to members of other subfamilies (e.g., lipases, dipeptidylaminopeptidase IV, etc.). To study the potentially moonlighting properties of alpha/beta hydrolases, the ESTHER database (for ESTerase and alpha/beta Hydrolase Enzymes and Relatives; http://bioweb.ensam.inra.fr/esther), which collects, organizes and disseminates structural and functional information related to alpha/beta hydrolases, has been updated with new tools and the web server interface has been upgraded. A new Overall Table along with a new Tree based on HMM models has been included to tentatively group subfamilies. These tools provide starting points for phylogenetic studies aimed at pinpointing the origin of duplications leading to paralogous genes (e.g., acetylcholinesterase versus butyrylcholinesterase, or neuroligin versus carboxylesterase). Another of our goals is to implement new tools to distinguish catalytically active enzymes from non-catalytic proteins in poorly studied or annotated subfamilies.

  13. Protein corona composition of gold nanoparticles/nanorods affects amyloid beta fibrillation process

    Science.gov (United States)

    Mirsadeghi, Somayeh; Dinarvand, Rassoul; Ghahremani, Mohammad Hossein; Hormozi-Nezhad, Mohammad Reza; Mahmoudi, Zohreh; Hajipour, Mohammad Javad; Atyabi, Fatemeh; Ghavami, Mahdi; Mahmoudi, Morteza

    2015-03-01

    Protein fibrillation process (e.g., from amyloid beta (Aβ) and α-synuclein) is the main cause of several catastrophic neurodegenerative diseases such as Alzheimer's and Parkinson diseases. During the past few decades, nanoparticles (NPs) were recognized as one of the most promising tools for inhibiting the progress of the disease by controlling the fibrillation kinetic process; for instance, gold NPs have a strong capability to inhibit Aβ fibrillations. It is now well understood that a layer of biomolecules would cover the surface of NPs (so called ``protein corona'') upon the interaction of NPs with protein sources. Due to the fact that the biological species (e.g., cells and amyloidal proteins) ``see'' the protein corona coated NPs rather than the pristine coated particles, one should monitor the fibrillation process of amyloidal proteins in the presence of corona coated NPs (and not pristine coated ones). Therefore, the previously obtained data on NPs effects on the fibrillation process should be modified to achieve a more reliable and predictable in vivo results. Herein, we probed the effects of various gold NPs (with different sizes and shapes) on the fibrillation process of Aβ in the presence and absence of protein sources (i.e., serum and plasma). We found that the protein corona formed a shell at the surface of gold NPs, regardless of their size and shape, reducing the access of Aβ to the gold inhibitory surface and, therefore, affecting the rate of Aβ fibril formation. More specifically, the anti-fibrillation potencies of various corona coated gold NPs were strongly dependent on the protein source and their concentrations (10% serum/plasma (simulation of an in vitro milieu) and 100% serum/plasma (simulation of an in vivo milieu)).Protein fibrillation process (e.g., from amyloid beta (Aβ) and α-synuclein) is the main cause of several catastrophic neurodegenerative diseases such as Alzheimer's and Parkinson diseases. During the past few decades

  14. Bovine latent transforming growth factor beta 1-binding protein 2: molecular cloning, identification of tissue isoforms, and immunolocalization to elastin-associated microfibrils.

    Science.gov (United States)

    Gibson, M A; Hatzinikolas, G; Davis, E C; Baker, E; Sutherland, G R; Mecham, R P

    1995-12-01

    Monoclonal antibodies to fibrillin 1 (MP340), a component of elastin-associated microfibrils, were used to screen cDNA libraries made from bovine nuchal ligament mRNA. One of the selected clones (cL9; 1.2 kb) hybridized on Northern (RNA) blotting with nuchal ligament mRNA to two abundant mRNAs of 9.0 and 7.5 kb, which were clearly distinct from fibrillin mRNA (10 kb). Further library screening and later reverse transcription PCR by the rapid amplification of cDNA ends (RACE) technique resulted in the isolation of additional overlapping cDNAs corresponding to about 6.7 kb of the mRNA. The encoded protein exhibited sequence similarity of around 80% with a recently identified human protein named latent transforming growth factor beta 1 (TGF-beta 1)-binding protein 2 (LTBP-2), indicating that the new protein was bovine LTBP-2. This was confirmed by the specific localization of bovine LTBP-2 cDNA probes to human chromosome 14q24.3, which is the locus of the human LTBP-2 gene. The domain structure of bovine LTBP-2 is very similar to that of the human LTBP-2, containing 20 examples of 6-cysteine epidermal growth factor-like repeats, 16 of which have the consensus sequence for calcium binding, together with 4 examples of 8-cysteine motifs characteristic of fibrillins and LTBP-1. A 4-cysteine sequence which is unique to bovine LTBP-2 and which has similarity to the 8-cysteine motifs was also present. Antibodies raised to two unique bovine LTBP-2 peptides specifically localized in tissue sections to the elastin-associated microfibrils, indicating that LTBP-2 is closely associated with these structures. Immunoblotting experiments identified putative LTBP-2 isoforms as a 260-kDa species released into the medium by cultured elastic tissue cells and as larger 290- and 310-kDa species in tissue extracts. A major proportion of tissue-derived LTBP-2 required treatment with 6 M guanidine for solubilization, indicating that the protein was strongly bound to the microfibrils. Most of

  15. EFEK SUPLEMENTASI EKSTRAK PROTEIN KECAMBAH KEDELAI TERHADAP KADAR IL-1BETA PENDERITA DIABETES TIPE-2 [The Effect of Soy Germ Protein Extract Supplementation on the Level of IL-1 Beta of Type-2 Diabetic Woman

    Directory of Open Access Journals (Sweden)

    Hery Winarsi1*

    2010-06-01

    Full Text Available The research was conducted to determine the effect of soy germ protein extract supplementation on the IL-1beta level of type-2 diabetic women. Research subjects were 32 type-2 diabetic women, outpatients of Diabetic Clinics of Margono Soekarjo General Hospital-Purwokerto. These women had blood glucose level above normal, BMI > 23 kg/m2, over 40 years old, lived in Purwokerto, and signed the informed consent. Group A (n=8 was given milk containing soy germ protein extract plus Zinc, Group B (n=8 was given milk containing soy germ protein extract without Zinc, Group C (n=8 was given placebo, and Group D (n=8 was given glibenclamide for 8 weeks. Blood samples ware taken 3 times: 0, 4 and 8 weeks after intervention. The plasma IL-1beta level was analyzed using Elisa. After 8 weeks, the IL-1beta level decreased from 6.01 pg/mL to 2.63 pg/mL (p=0.022 in group A. However, the IL-1beta level in Group A was not different from the Group B (p=0.51. Soy germ protein extract decreased cytokine inflammatory production of type-2 dibetic. Soy germ protein extract with or without Zn had similar effects to glibenclamide in reducing the IL-1beta level (p=0.76.

  16. A beta-barrel outer membrane protein facilitates cellular uptake of polychlorophenols in Cupriavidus necator.

    Science.gov (United States)

    Belchik, Sara Mae; Schaeffer, Scott M; Hasenoehrl, Shelley; Xun, Luying

    2010-06-01

    The tcpRXABCYD operon of Cupriavidus necator JMP134 is involved in the degradation of 2,4,6-trichlorophenol (TCP). All of the gene products except TcpY have assigned functions in TCP metabolism. Sequence comparison identified TcpY as a member of COG4313, a group of hypothetical proteins. TcpY has a signal peptide, indicating it is a membrane or secreted protein. Secondary structure and topology analysis indicated TcpY as a beta-barrel outer membrane protein, similar to the Escherichia coli outer membrane protein FadL that transports hydrophobic long-chain fatty acids. Constitutive expression of tcpY in two C. necator strains rendered the cells more sensitive to TCP and other polychlorophenols. Further, C. necator JMP134 expressing cloned tcpY transported more TCP into the cell than a control with the cloning vector. Thus, TcpY is an outer membrane protein that facilitates the passing of polychlorophenols across the outer membrane of C. necator. Similarly, other COG4313 proteins are possibly outer membrane transporters of hydrophobic aromatic compounds.

  17. Genus beta human papillomavirus E6 proteins vary in their effects on the transactivation of p53 target genes.

    Science.gov (United States)

    White, Elizabeth A; Walther, Johanna; Javanbakht, Hassan; Howley, Peter M

    2014-08-01

    The genus beta human papillomaviruses (beta HPVs) cause cutaneous lesions and are thought to be involved in the initiation of some nonmelanoma skin cancers (NMSCs), particularly in patients with the genetic disorder epidermodysplasia verruciformis (EV). We have previously reported that at least two of the genus beta HPV E6 proteins bind to and/or increase the steady-state levels of p53 in squamous epithelial cells. This is in contrast to a well-characterized ability of the E6 proteins of cancer-associated HPVs of genus alpha HPV, which inactivate p53 by targeting its ubiquitin-mediated proteolysis. In this study, we have investigated the ability of genus beta E6 proteins from eight different HPV types to block the transactivation of p53 target genes following DNA damage. We find that the E6 proteins from diverse beta HPV species and types vary in their capacity to block the induction of MDM2, p21, and proapoptotic genes after genotoxic stress. We conclude that some genus beta HPV E6 proteins inhibit at least some p53 target genes, although perhaps not by the same mechanism or to the same degree as the high-risk genus alpha HPV E6 proteins. This study addresses the ability of various human papillomavirus E6 proteins to block the activation of p53-responsive cellular genes following DNA damage in human keratinocytes, the normal host cell for HPVs. The E6 proteins encoded by the high-risk, cancer-associated HPV types of genus alpha HPV have a well-established activity to target p53 degradation and thereby inhibit the response to DNA damage. In this study, we have investigated the ability of genus beta HPV E6 proteins from eight different HPV types to block the ability of p53 to transactivate downstream genes following DNA damage. We find that some, but not all, genus beta HPV E6 proteins can block the transactivation of some p53 target genes. This differential response to DNA damage furthers the understanding of cutaneous HPV biology and may help to explain the

  18. Expression of TGF-β2 mRNA and PCNA, FN Protein in Lens Epithelial Cells in Age-related Nuclear and Cortex Cataract

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    By using RT-PCR and immunohistochemistry, the expressions of transforming growth factor β2 (TGF-β2) mRNA, proliferating cell nuclear antigen (PCNA) and fibronection (FN) protein in lens epithelial cells (LECs) of age-related nuclear and cortex cataract were detected and compared. The results of RT-PCR revealed that the expression of TGF-β2 mRNA was higher in cortex cataract than in nuclear cataract. Immunohistochemistry demonstrated that the expression of PCNA protein was lower and the expression of FN protein was higher in cortex cataract than in nuclear cataract. It was suggested that TGF-β2, PCNA and FN might take important parts in the process of age-related cataract. Cortex cataract was related to the transdifferentiation of LECs, and nuclear cataract to the proliferation of LECs.

  19. Fragile X mental retardation protein interactions with a G quadruplex structure in the 3'-untranslated region of NR2B mRNA.

    Science.gov (United States)

    Stefanovic, Snezana; DeMarco, Brett A; Underwood, Ayana; Williams, Kathryn R; Bassell, Gary J; Mihailescu, Mihaela Rita

    2015-12-01

    Fragile X syndrome, the most common cause of inherited intellectual disability, is caused by a trinucleotide CGG expansion in the 5'-untranslated region of the FMR1 gene, which leads to the loss of expression of the fragile X mental retardation protein (FMRP). FMRP, an RNA-binding protein that regulates the translation of specific mRNAs, has been shown to bind a subset of its mRNA targets by recognizing G quadruplex structures. It has been suggested that FMRP controls the local protein synthesis of several protein components of the post synaptic density (PSD) in response to specific cellular needs. We have previously shown that the interactions between FMRP and mRNAs of the PSD scaffold proteins PSD-95 and Shank1 are mediated via stable G-quadruplex structures formed within the 3'-untranslated regions of these mRNAs. In this study we used biophysical methods to show that a comparable G quadruplex structure forms in the 3'-untranslated region of the glutamate receptor subunit NR2B mRNA encoding for a subunit of N-methyl-d-aspartate (NMDA) receptors that is recognized specifically by FMRP, suggesting a common theme for FMRP recognition of its dendritic mRNA targets.

  20. Effects of estradiol-17beta administration on steady-state messenger ribonucleic acid (MRNA) encoding equine alpha and LH/CGbeta subunits in pituitaries of ovariectomized pony mares.

    Science.gov (United States)

    Sharp, D C; Wolfe, M W; Cleaver, B D; Nilson, J

    2001-03-15

    The process of sexual recrudescence in the springtime in mares is characterized by renewal of follicular growth and acquisition of steroidogenic competence. Concomitant with renewal of follicular steroidogenesis is re-establishment of LH biosynthesis and secretion. Research results from our laboratory indicate that increased estradiol and LH secretion occur in close temporal association before the first ovulation of the year. Therefore, the hypothesis tested in this experiment was that estrogen administration to ovariectomized pony mares during the equivalent time of early vernal transition would enhance LH biosynthesis as monitored by messenger ribonucleic acid (mRNA) encoding for the pituitary subunits of LH (alpha and LH/CGbeta). Mares were administered either sesame oil vehicle control, or estradiol (5 mg i.m. twice daily in sesame oil) for 3, 6 or 9 days, beginning on February 2. The pituitary glands were harvested, and examined for LH subunit mRNA by Northern Blot and slot blot analysis. There was a significant increase in LH secretion after 6 days of estradiol secretion compared with control vehicle administration. Similarly, there was a significant increase in both alpha and LH/CGbeta subunit mRNA when estradiol was administered for 9 days. These data indicate that estrogen stimulates LH subunit formation in mares during early equivalent vernal transition. These data do not, however, discriminate between a direct pituitary effect of estrogen, and a hypothalamic effect. Whether the surge of estradiol just prior to the first ovulation of the year is essential for the renewed biosynthesis of LH subunits cannot be determined from these data. However an important role of estrogen in the final stages of sexual recrudescence is indicated.

  1. [The relationship between the effects of MTA on mRNA expression of four iconic proteins in cells of fetal rat skull and cell culture environment].

    Science.gov (United States)

    Zheng, Jun-Yuan; He, Li; Hu, Tu-qiang

    2016-02-01

    To discuss the influence of MTA on mRNA expression of Cbfa1, ALP, Col-Ⅰand BGP which are 4 kinds of iconic protein in cells of fetal rats skull, and explore its influence on cell culture environment and association of changes of calcium, phosphorus. Cells were obtained by 2 kinds of mixed enzymatic digestion for 3 steps from gestation fetal rat calvarial bone. The expression of Cbfa1mRNA, ALPmRNA, Col-1mRNA, BGP mRNA and extracellular calcium were detected. Phosphorus (P) and calcium concentration of fetal rat skull cells co-cultured with MTA for 3 weeks at different stages of cell differentiation was assessed atomic absorption spectrophotometry. The data was statistically analyzed using SPSS10.0 software package. At the 4th day, P3- content decreased significantly (PMTA group increased most greatly and was 40 times of the control group. At the 14th to 18th day, the Ca2+ and P3- content reduced significantly (PMTA group rised most greatly which was about 7.71 times of the control group. Then Cbfalpha l mRNA in MTA group increased most strongly later which was about 7.38 times of the control group. Col Ⅰ mRNA increased minimally in all time points. The change of P3- content may be the initiating factor when MTA promoted differentiation of fetal rats skull cells in vitro, and Ca2+ could greatly accelerate the process of mineralization when accumulated to a certain extent. At the same time, the expression of ALPmRNA, BGPmRNA, Col Ⅰ mRNA and Cbfalpha lmRNA were regulated accordingly, which is the key to explain osteogenetic mechanism of MTA.

  2. The Minor Allele of rs7574865 in the STAT4 Gene Is Associated with Increased mRNA and Protein Expression

    Science.gov (United States)

    Lamana, Amalia; López-Santalla, Mercedes; Castillo-González, Raquel; Ortiz, Ana María; Martín, Javier; García-Vicuña, Rosario; González-Álvaro, Isidoro

    2015-01-01

    Objective The T allele of rs7574865 in STAT4 confers risk of developing autoimmune disorders. However, its functional significance remains unclear. Here we analyze how rs7574865 affects the transcription of STAT4 and its protein expression. Methods We studied 201 patients (80% female; median age, 54 years; median disease duration, 5.4 months) from PEARL study. Demographic, clinical, laboratory and therapeutic data were collected at each visit. IL-6 serum levels were measured by enzyme immune assay. The rs7574865 was genotyped using TaqMan probes. The expression levels of STAT4 mRNA were determined at 182 visits from 69 patients using quantitative real-time polymerase chain reaction. STAT4 protein was assessed by western blot in 62 samples from 34 patients. To determine the effect of different variables on the expression of STAT4 mRNA and protein, we performed multivariate longitudinal analyses using generalized linear models. Results After adjustment for age, disease activity and glucocorticoid dose as confounders, the presence of at least one copy of the T allele of rs7574865 was significantly associated with higher levels of STAT4 mRNA. Similarly, TT patients showed significantly higher levels of STAT4 protein than GG patients. IL-6 induced STAT4 and STAT5 phosphorylation in peripheral blood lymphocytes. Patients carrying at least one T allele of rs7574865 displayed lower levels of serum IL-6 compared to GG homozygous; by contrast the production of C-reactive protein was similar in both populations. Conclusion Our data suggest that the presence of the rs7574865 T allele enhances STAT4 mRNA transcription and protein expression. It may enhance the signaling of molecules depending on the STAT4 pathway. PMID:26569609

  3. Saturated fatty acids induce post-transcriptional regulation of HAMP mRNA via AU-rich element-binding protein, human antigen R (HuR).

    Science.gov (United States)

    Lu, Sizhao; Mott, Justin L; Harrison-Findik, Duygu Dee

    2015-10-02

    Iron is implicated in fatty liver disease pathogenesis. The human hepcidin gene, HAMP, is the master switch of iron metabolism. The aim of this study is to investigate the regulation of HAMP expression by fatty acids in HepG2 cells. For these studies, both saturated fatty acids (palmitic acid (PA) and stearic acid (SA)) and unsaturated fatty acid (oleic acid (OA)) were used. PA and, to a lesser extent, SA, but not OA, up-regulated HAMP mRNA levels, as determined by real-time PCR. To understand whether PA regulates HAMP mRNA at the transcriptional or post-transcriptional level, the transcription inhibitor actinomycin D was employed. PA-mediated induction of HAMP mRNA expression was not blocked by actinomycin D. Furthermore, PA activated HAMP 3'-UTR, but not promoter, activity, as shown by reporter assays. HAMP 3'-UTR harbors a single AU-rich element (ARE). Mutation of this ARE abolished the effect of PA, suggesting the involvement of ARE-binding proteins. The ARE-binding protein human antigen R (HuR) stabilizes mRNA through direct interaction with AREs on 3'-UTR. HuR is regulated by phosphorylation-mediated nucleo-cytoplasmic shuttling. PA activated this process. The binding of HuR to HAMP mRNA was also induced by PA in HepG2 cells. Silencing of HuR by siRNA abolished PA-mediated up-regulation of HAMP mRNA levels. PKC is known to phosphorylate HuR. Staurosporine, a broad-spectrum PKC inhibitor, inhibited both PA-mediated translocation of HuR and induction of HAMP expression. Similarly, rottlerin, a novel class PKC inhibitor, abrogated PA-mediated up-regulation of HAMP expression. In conclusion, lipids mediate post-transcriptional regulation of HAMP throughPKC- and HuR-dependent mechanisms.

  4. Galectin-3 is a non-classic RNA binding protein that stabilizes the mucin MUC4 mRNA in the cytoplasm of cancer cells

    Science.gov (United States)

    Coppin, Lucie; Vincent, Audrey; Frénois, Frédéric; Duchêne, Belinda; Lahdaoui, Fatima; Stechly, Laurence; Renaud, Florence; Villenet, Céline; Seuningen, Isabelle Van; Leteurtre, Emmanuelle; Dion, Johann; Grandjean, Cyrille; Poirier, Françoise; Figeac, Martin; Delacour, Delphine; Porchet, Nicole; Pigny, Pascal

    2017-01-01

    Pancreatic cancer cells express high levels of MUC1, MUC4 and MUC16 mRNAs that encode membrane-bound mucins. These mRNAs share unusual features such as a long half-life. However, it remains unknown how mucin mRNA stability is regulated. Galectin-3 (Gal-3) is an endogenous lectin playing important biological functions in epithelial cells. Gal-3 is encoded by LGALS3 which is up-regulated in pancreatic cancer. Despite the absence of a RNA-recognition motif, Gal-3 interacts indirectly with pre-mRNAs in the nucleus and promotes constitutive splicing. However a broader role of Gal-3 in mRNA fate is unexplored. We report herein that Gal-3 increases MUC4 mRNA stability through an intermediate, hnRNP-L which binds to a conserved CA repeat element in the 3′UTR in a Gal-3 dependent manner and also controls Muc4 mRNA levels in epithelial tissues of Gal3−/− mice. Gal-3 interacts with hnRNP-L in the cytoplasm, especially during cell mitosis, but only partly associates with protein markers of P-Bodies or Stress Granules. By RNA-IP plus RNA-seq analysis and imaging, we demonstrate that Gal-3 binds to mature spliced MUC4 mRNA in the perinuclear region, probably in hnRNP-L-containing RNA granules. Our findings highlight a new role for Gal-3 as a non-classic RNA-binding protein that regulates MUC4 mRNA post-transcriptionally. PMID:28262838

  5. IL-1beta induced protein changes in diabetes prone BB rat islets of Langerhans identified by proteome analysis

    DEFF Research Database (Denmark)

    Sparre, T; Bjerre-Christensen, Ulla; Mose Larsen, P;

    2002-01-01

    of 82 out of 1 815 protein spots detected by two dimensional gel electrophoresis in IL-1beta exposed diabetes prone Bio Breeding (BB-DP) rat islets of Langerhans in vitro. The aim of this study was to identify the proteins in these 82 spots by mass spectrometry and compare these changes with those seen...

  6. Cellular uptake of beta-carotene from protein stabilized solid lipid nano-particles prepared by homogenization-evaporation method

    Science.gov (United States)

    Using a homogenization-evaporation method, beta-carotene (BC) loaded nano-particles were prepared with different ratios of food-grade sodium caseinate (SC), whey protein isolate (WPI), or soy protein isolate (SPI) to BC and evaluated for their physiochemical stability, in vitro cytotoxicity, and cel...

  7. CCAAT/enhancer binding protein {beta} deletion increases mitochondrial function and protects mice from LXR-induced hepatic steatosis

    Energy Technology Data Exchange (ETDEWEB)

    Rahman, Shaikh M., E-mail: rmizanoor@hotmail.com [Department of Pediatrics, School of Medicine, University of Colorado Denver, Aurora, CO 80045 (United States); Choudhury, Mahua; Janssen, Rachel C.; Baquero, Karalee C. [Department of Pediatrics, School of Medicine, University of Colorado Denver, Aurora, CO 80045 (United States); Miyazaki, Makoto [Division of Renal Diseases and Hypertension, School of Medicine, University of Colorado Denver, Aurora, CO 80045 (United States); Friedman, Jacob E. [Department of Pediatrics, School of Medicine, University of Colorado Denver, Aurora, CO 80045 (United States); Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Denver, Aurora, CO 80045 (United States)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer LXR agonist activation increases liver TG accumulation by increasing lipogenesis. Black-Right-Pointing-Pointer C/EBP{beta}{sup -/-} mouse prevents LXR activation-mediated induction of hepatic lipogenesis. Black-Right-Pointing-Pointer C/EBP{beta} deletion increases mitochondrial transport chain function. Black-Right-Pointing-Pointer Beneficial effects of LXR activation on liver cholesterol metabolism did not change. Black-Right-Pointing-Pointer C/EBP{beta} inhibition might have important therapeutic potential. -- Abstract: Drugs designed specifically to activate liver X receptors (LXRs) have beneficial effects on lowering cholesterol metabolism and inflammation but unfortunately lead to severe hepatic steatosis. The transcription factor CCAAT/enhancer binding protein beta (C/EBP{beta}) is an important regulator of liver gene expression but little is known about its involvement in LXR-based steatosis and cholesterol metabolism. The present study investigated the role of C/EBP{beta} expression in LXR agonist (T0901317)-mediated alteration of hepatic triglyceride (TG) and lipogenesis in mice. C/EBP{beta} deletion in mice prevented LXR agonist-mediated induction of lipogenic gene expression in liver in conjunction with significant reduction of liver TG accumulation. Surprisingly, C/EBP{beta}{sup -/-} mice showed a major increase in liver mitochondrial electron chain function compared to WT mice. Furthermore, LXR activation in C/EBP{beta}{sup -/-} mice increased the expression of liver ATP-binding cassette transporter ABCG1, a gene implicated in cholesterol efflux and reducing blood levels of total and LDL-cholesterol. Together, these findings establish a central role for C/EBP{beta} in the LXR-mediated steatosis and mitochondrial function, without impairing the influence of LXR activation on lowering LDL and increasing HDL-cholesterol. Inactivation of C/EBP{beta} might therefore be an important therapeutic strategy to prevent LXR

  8. The structurally disordered paramyxovirus nucleocapsid protein tail domain is a regulator of the mRNA transcription gradient

    Science.gov (United States)

    Cox, Robert M.; Krumm, Stefanie A.; Thakkar, Vidhi D.; Sohn, Maximilian; Plemper, Richard K.

    2017-01-01

    The paramyxovirus RNA-dependent RNA-polymerase (RdRp) complex loads onto the nucleocapsid protein (N)–encapsidated viral N:RNA genome for RNA synthesis. Binding of the RdRp of measles virus (MeV), a paramyxovirus archetype, is mediated through interaction with a molecular recognition element (MoRE) located near the end of the carboxyl-terminal Ntail domain. The structurally disordered central Ntail section is thought to add positional flexibility to MoRE, but the functional importance of this Ntail region for RNA polymerization is unclear. To address this question, we dissected functional elements of Ntail by relocating MoRE into the RNA-encapsidating Ncore domain. Linker-scanning mutagenesis identified a microdomain in Ncore that tolerates insertions. MoRE relocated to Ncore supported efficient interaction with N, MoRE-deficient Ntails had a dominant-negative effect on bioactivity that was alleviated by insertion of MoRE into Ncore, and recombinant MeV encoding N with relocated MoRE grew efficiently and remained capable of mRNA editing. MoRE in Ncore also restored viability of a recombinant lacking the disordered central Ntail section, but this recombinant was temperature-sensitive, with reduced RdRp loading efficiency and a flattened transcription gradient. These results demonstrate that virus replication requires high-affinity RdRp binding sites in N:RNA, but productive RdRp binding is independent of positional flexibility of MoRE and cis-acting elements in Ntail. Rather, the disordered central Ntail section independent of the presence of MoRE in Ntail steepens the paramyxovirus transcription gradient by promoting RdRp loading and preventing the formation of nonproductive polycistronic viral mRNAs. Disordered Ntails may have evolved as a regulatory element to adjust paramyxovirus gene expression. PMID:28168220

  9. The Expression of Interleukin-17, Interferon-gamma, and Macrophage Inflammatory Protein-3 Alpha mRNA in Patients with Psoriasis Vulgaris

    Institute of Scientific and Technical Information of China (English)

    李家文; 李东升; 谭志建

    2004-01-01

    Summary: To investigate the role of Interleukin-17 (IL-17), Interferon-gamma (IFN-γ), and macrophage inflammatory protein-3 alpha (MIP-3α) in the pathogenesis of psoriasis, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to semi-quantitatively analyze the mRNA expression of IL 17, IFN-γ, and MIP-3α in 31 psoriatic lesions and 16 normal skin tissues. The results showed that the mRNA of the three cytokines was present in all specimens. And the expression level of IL-17 mRNA in skin lesions was 1. 1416±0. 0591, which was significantly higher than that in normal controls (0. 8788±0. 0344, P<0. 001). The expression levels of IFN-γ mRNA were 1.1142±0. 0561 and 0. 9050±0. 0263, respectively, with significant difference(P<0. 001). And the expression levels of MIP-3α mRNA in psoriatic lesions was 1. 1397 ± 0. 0521, which was markedly higher than that in normal controls (0. 8681±0. 0308, P<0. 001). These findings indicate that up regulated expression of IL-17, IFN-γ, and MIP-3α might be involved in the pathogenesis of psoriasis.

  10. Legg-Calvé-Perthes disease, protein C deficiency, and beta-thalassemia major: report of two cases.

    Science.gov (United States)

    Levin, C; Zalman, L; Shalev, S; Mader, R; Koren, A

    2000-01-01

    Legg-Calvé-Perthes disease is an idiopathic osteonecrosis or avascular necrosis of the capital femoral epiphysis and the associated complications thereof occurring in an immature growing child. The association between osteonecrosis of the femoral head and thrombophilia was postulated by Glueck in 1994. We describe Legg-Calvé-Perthes disease associated with protein C deficiency and beta-thalassemia major in two children among a cohort of 79 beta-thalassemia patients treated in our clinic. The association of thrombophilia, aseptic necrosis of the femoral head, and beta-thalassemia has not been previously described in the literature.

  11. Inhibition of protein tyrosine phosphatase 1beta by hispidin derivatives isolated from the fruiting body of Phellinus linteus.

    Science.gov (United States)

    Lee, Yeon Sil; Kang, Il-Jun; Won, Moo Ho; Lee, Jae-Yong; Kim, Jin Kyu; Lim, Soon Sung

    2010-12-01

    Protein tyrosine phosphatase 1beta (PTP1beta) acts as a negative regulator of insulin signaling. Selective inhibition of PTP1beta has served as a potential drug target for the treatment of type 2 diabetes mellitus. We evaluated the inhibitory effect of Phellinus linteus against PTP1beta as part of our ongoing search for natural therapeutic and preventive agents for diabetes mellitus. Fractions of the P. linteus extract were found to exhibit significant inhibitory activities against PTP1beta. In an attempt to identify bioactive components, we isolated, from the most active ethyl acetate fraction, five hispidin derivatives (phelligridimer A, davallialactone, hypholomine B, interfungins A, and inoscavin A) and four phenolic compounds (protocatechuic acid, protocatechualdehyde, caffeic acid, and ellagic acid). The chemical structures of these compounds were elucidated from spectroscopic evidence and by comparison with published data. All the compounds strongly inhibited PTP1beta activity in an in vitro assay; their IC50 values ranged from 9.0 +/- 0.01 to 58.2 +/- 0.3 microM. Our results indicated that the hispidin skeleton may be an important moiety for inhibitory activity of the above compounds against PTP1beta. Thus, hispidin derivatives could be a potent new class of natural PTP1beta inhibitors.

  12. Differential role of Sloan-Kettering Institute (Ski) protein in Nodal and transforming growth factor-beta (TGF-β)-induced Smad signaling in prostate cancer cells.

    Science.gov (United States)

    Vo, BaoHan T; Cody, Bianca; Cao, Yang; Khan, Shafiq A

    2012-11-01

    Transforming growth factor-beta (TGF-β) signaling pathways contain both tumor suppressor and tumor promoting activities. We have demonstrated that Nodal, another member of the TGF-β superfamily, and its receptors are expressed in prostate cancer cells. Nodal and TGF-β exerted similar biological effects on prostate cells; both inhibited proliferation in WPE, RWPE1 and DU145 cells, whereas neither had any effect on the proliferation of LNCaP or PC3 cells. Interestingly, Nodal and TGF-β induced migration in PC3 cells, but not in DU145 cells. TGF-β induced predominantly phosphorylation of Smad3, whereas Nodal induced phosphorylation of only Smad2. We also determined the expression and differential role of Ski, a corepressor of Smad2/3, in Nodal and TGF-β signaling in prostate cancer cells. Similar levels of Ski mRNA were found in several established prostate cell lines; however, high levels of Ski protein were only detected in prostate cancer cells and prostate cancer tissue samples. Exogenous Nodal and TGF-β had no effects on Ski mRNA levels. On the other hand, TGF-β induced a rapid degradation of Ski protein mediated by the proteasomal pathway, whereas Nodal had no effect on Ski protein. Reduced Ski levels correlated with increased basal and TGF-β-induced Smad2/3 phosphorylation. Knockdown of endogenous Ski reduced proliferation in DU145 cells and enhanced migration of PC3 cells. We conclude that high levels of Ski expression in prostate cancer cells may be responsible for repression of TGF-β and Smad3 signaling, but Ski protein levels do not influence Nodal and Smad2 signaling.

  13. Heat shock protein-90-beta facilitates enterovirus 71 viral particles assembly.

    Science.gov (United States)

    Wang, Robert Y L; Kuo, Rei-Lin; Ma, Wei-Chieh; Huang, Hsing-I; Yu, Jau-Song; Yen, Sih-Min; Huang, Chi-Ruei; Shih, Shin-Ru

    2013-09-01

    Molecular chaperones are reported to be crucial for virus propagation, but are not yet addressed in Human Enterovirus 71 (EV71). Here we describe the specific association of heat shock protein-90-beta (Hsp90β), but not alpha form (Hsp90α), with EV71 viral particles by the co-purification with virions using sucrose density gradient ultracentrifugation, and by the colocalization with viral particles, as assessed by immunogold electron microscopy. The reduction of the Hsp90β protein using RNA interference decreased the correct assembly of viral particles, without affecting EV71 replication levels. Tracking ectopically expressed Hsp90β protein associated with EV71 virions revealed that Hsp90β protein was transmitted to new host cells through its direct association with infectious viral particles. Our findings suggest a new antiviral strategy in which extracellular Hsp90β protein is targeted to decrease the infectivity of EV71 and other enteroviruses, without affecting the broader functions of this constitutively expressed molecular chaperone.

  14. Biological activities of the antiviral protein BE27 from sugar beet (Beta vulgaris L.).

    Science.gov (United States)

    Iglesias, Rosario; Citores, Lucía; Di Maro, Antimo; Ferreras, José M

    2015-02-01

    The ribosome inactivating protein BE27 displays several biological activities in vitro that could result in a broad action against several types of pathogens. Beetin 27 (BE27), a ribosome-inactivating protein (RIP) from sugar beet (Beta vulgaris L.) leaves, is an antiviral protein induced by virus and signaling compounds such as hydrogen peroxide and salicylic acid. Its role as a defense protein has been attributed to its RNA polynucleotide:adenosine glycosidase activity. Here we tested other putative activities of BE27 that could have a defensive role against pathogens finding that BE27 displays rRNA N-glycosidase activity against yeast and Agrobacterium tumefaciens ribosomes, DNA polynucleotide:adenosine glycosidase activity against herring sperm DNA, and magnesium-dependent endonuclease activity against the supercoiled plasmid PUC19 (nicking activity). The nicking activity could be a consequence of an unusual conformation of the BE27 active site, similar to that of PD-L1, a RIP from Phytolacca dioica L. leaves. Additionally, BE27 possesses superoxide dismutase activity, thus being able to produce the signal compound hydrogen peroxide. BE27 is also toxic to COLO 320 cells, inducing apoptosis in these cells by either activating the caspase pathways and/or inhibiting protein synthesis. The combined effect of these biological activities could result in a broad action against several types of pathogens such as virus, bacteria, fungi or insects.

  15. Heat shock protein-90-beta facilitates enterovirus 71 viral particles assembly

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Robert Y.L., E-mail: yuwang@mail.cgu.edu.tw [Research Center for Emerging Viral Infections, College of Medicine, Chang Gung University, Tao-Yuan 333, Taiwan (China); Department of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan 333 Taiwan (China); Kuo, Rei-Lin [Research Center for Emerging Viral Infections, College of Medicine, Chang Gung University, Tao-Yuan 333, Taiwan (China); Department of Medical Biotechnology and Laboratory Science and Graduate Program of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan 333, Taiwan (China); Ma, Wei-Chieh [Department of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan 333 Taiwan (China); Huang, Hsing-I [Research Center for Emerging Viral Infections, College of Medicine, Chang Gung University, Tao-Yuan 333, Taiwan (China); Department of Medical Biotechnology and Laboratory Science and Graduate Program of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan 333, Taiwan (China); Yu, Jau-Song [Department of Cell and Molecular Biology, College of Medicine, Chang Gung University, Tao-Yuan 333, Taiwan (China); Molecular Medicine Research Center, Chang Gung University, Tao-Yuan 333, Taiwan (China); Yen, Sih-Min [Department of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan 333 Taiwan (China); Huang, Chi-Ruei [Research Center for Emerging Viral Infections, College of Medicine, Chang Gung University, Tao-Yuan 333, Taiwan (China); Department of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan 333 Taiwan (China); Shih, Shin-Ru [Research Center for Emerging Viral Infections, College of Medicine, Chang Gung University, Tao-Yuan 333, Taiwan (China); Department of Medical Biotechnology and Laboratory Science and Graduate Program of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan 333, Taiwan (China)

    2013-09-01

    Molecular chaperones are reported to be crucial for virus propagation, but are not yet addressed in Human Enterovirus 71 (EV71). Here we describe the specific association of heat shock protein-90-beta (Hsp90β), but not alpha form (Hsp90α), with EV71 viral particles by the co-purification with virions using sucrose density gradient ultracentrifugation, and by the colocalization with viral particles, as assessed by immunogold electron microscopy. The reduction of the Hsp90β protein using RNA interference decreased the correct assembly of viral particles, without affecting EV71 replication levels. Tracking ectopically expressed Hsp90β protein associated with EV71 virions revealed that Hsp90β protein was transmitted to new host cells through its direct association with infectious viral particles. Our findings suggest a new antiviral strategy in which extracellular Hsp90β protein is targeted to decrease the infectivity of EV71 and other enteroviruses, without affecting the broader functions of this constitutively expressed molecular chaperone. - Highlights: • Hsp90β is associated with EV71 virion and is secreted with the release virus. • Hsp90β effects on the correct assembly of viral particles. • Viral titer of cultured medium was reduced in the presence of geldanamycin. • Viral titer was also reduced when Hsp90β was suppressed by siRNA treatment. • The extracellular Hsp90β was also observed in other RNA viruses-infected cells.

  16. Overexpression of estrogen receptor beta alleviates the toxic effects of beta-amyloid protein on PC12 cells via non-hormonal ligands

    Institute of Scientific and Technical Information of China (English)

    Hui Wang; Lihui Si; Xiaoxi Li; Weiguo Deng; Haimiao Yang; Yuyan Yang; Yan Fu

    2012-01-01

    After binding to the estrogen receptor, estrogen can alleviate the toxic effects of beta-amyloid protein, and thereby exert a therapeutic effect on Alzheimer's disease patients. Estrogen can increase the incidence of breast carcinoma and endometrial cancer in post-menopausal women, so it is not suitable for clinical treatment of Alzheimer's disease. There is recent evidence that the estrogen receptor can exert its neuroprotective effects without estrogen dependence. Real-time quantitative PCR and flow cytometry results showed that, compared with non-transfected PC12 cells, adenovirus-mediated estrogen receptor β gene-transfected PC12 cells exhibited lower expression of tumor necrosis factor α and interleukin 1β under stimulation with beta-amyloid protein and stronger protection from apoptosis. The Akt-specific inhibitor Abi-2 decreased the anti-inflammatory and anti-apoptotic effects of estrogen receptor β gene-transfection. These findings suggest that overexpression of estrogen receptor β can alleviate the toxic effect of beta-amyloid protein on PC12 cells, without estrogen dependence. The Akt pathway is one of the potential means for the anti-inflammatory and anti-apoptotic effects of the estrogen receptor.

  17. Mechanism of neuronal versus endothelial cell uptake of Alzheimer's disease amyloid beta protein.

    Directory of Open Access Journals (Sweden)

    Karunya K Kandimalla

    Full Text Available Alzheimer's disease (AD is characterized by significant neurodegeneration in the cortex and hippocampus; intraneuronal tangles of hyperphosphorylated tau protein; and accumulation of beta-amyloid (Abeta proteins 40 and 42 in the brain parenchyma as well as in the cerebral vasculature. The current understanding that AD is initiated by the neuronal accumulation of Abeta proteins due to their inefficient clearance at the blood-brain-barrier (BBB, places the neurovascular unit at the epicenter of AD pathophysiology. The objective of this study is to investigate cellular mechanisms mediating the internalization of Abeta proteins in the principle constituents of the neurovascular unit, neurons and BBB endothelial cells. Laser confocal micrographs of wild type (WT mouse brain slices treated with fluorescein labeled Abeta40 (F-Abeta40 demonstrated selective accumulation of the protein in a subpopulation of cortical and hippocampal neurons via nonsaturable, energy independent, and nonendocytotic pathways. This groundbreaking finding, which challenges the conventional belief that Abeta proteins are internalized by neurons via receptor mediated endocytosis, was verified in differentiated PC12 cells and rat primary hippocampal (RPH neurons through laser confocal microscopy and flow cytometry studies. Microscopy studies have demonstrated that a significant proportion of F-Abeta40 or F-Abeta42 internalized by differentiated PC12 cells or RPH neurons is located outside of the endosomal or lysosomal compartments, which may accumulate without degradation. In contrast, BBME cells exhibit energy dependent uptake of F-Abeta40, and accumulate the protein in acidic cell organelle, indicative of endocytotic uptake. Such a phenomenal difference in the internalization of Abeta40 between neurons and BBB endothelial cells may provide essential clues to understanding how various cells can differentially regulate Abeta proteins and help explain the vulnerability of cortical

  18. Sequence-specific binding of a hormonally regulated mRNA binding protein to cytidine-rich sequences in the lutropin receptor open reading frame.

    Science.gov (United States)

    Kash, J C; Menon, K M

    1999-12-21

    In previous studies, a lutropin receptor mRNA binding protein implicated in the hormonal regulation of lutropin receptor mRNA stability was identified. This protein, termed LRBP-1, was shown by RNA gel electrophoretic mobility shift assay to specifically interact with lutropin receptor RNA sequences. The present studies have examined the specificity of lutropin receptor mRNA recognition by LRBP-1 and mapped the contact site by RNA footprinting and by site-directed mutagenesis. LRBP-1 was partially purified by cation-exchange chromatography, and the mRNA binding properties of the partially purified LRBP-1 were examined by RNA gel electrophoretic mobility shift assay and hydroxyl-radical RNA footprinting. These data showed that the LRBP-1 binding site is located between nucleotides 203 and 220 of the receptor open reading frame, and consists of the bipartite polypyrimidine sequence 5'-UCUC-X(7)-UCUCCCU-3'. Competition RNA gel electrophoretic mobility shift assays demonstrated that homoribopolymers of poly(rC) were effective RNA binding competitors, while poly(rA), poly(rG), and poly(rU) showed no effect. Mutagenesis of the cytidine residues contained within the LRBP-1 binding site demonstrated that all the cytidines in the bipartite sequence contribute to LRBP-1 binding specificity. Additionally, RNA gel electrophoretic mobility supershift analysis showed that LRBP-1 was not recognized by antibodies against two well-characterized poly(rC) RNA binding proteins, alphaCP-1 and alphaCP-2, implicated in the regulation of RNA stability of alpha-globin and tyrosine hydroxylase mRNAs. In summary, we show that partially purified LRBP-1 binds to a polypyrimidine sequence within nucleotides 203 and 220 of lutropin receptor mRNA with a high degree of specificity which is indicative of its role in posttranscriptional control of lutropin receptor expression.

  19. Enhanced expression of the type II transforming growth factor beta receptor in human pancreatic cancer cells without alteration of type III receptor expression.

    Science.gov (United States)

    Friess, H; Yamanaka, Y; Büchler, M; Berger, H G; Kobrin, M S; Baldwin, R L; Korc, M

    1993-06-15

    We have recently found that human pancreatic adenocarcinomas exhibit strong immunostaining for the three mammalian transforming growth factor beta (TGF-beta) isoforms. These important growth-regulating polypeptides bind to a number of proteins, including the type I TGF-beta receptor (T beta R-I), type II TGF-beta receptor (T beta R-II), and the type III TGF-beta receptor (T beta R-III). In the present study we sought to determine whether T beta R-II and T beta R-III expression is altered in pancreatic cancer. Northern blot analysis indicated that, by comparison with the normal pancreas, pancreatic adenocarcinomas exhibited a 4.6-fold increase (P beta R-II. In contrast, mRNA levels encoding T beta R-III were not increased. In situ hybridization showed that T beta R-II mRNA was expressed in the majority of cancer cells, whereas mRNA grains encoding T beta R-III were detectable in only a few cancer cells and were present mainly in the surrounding stroma. These findings suggest that enhanced levels of T beta R-II may have a role in regulating human pancreatic cancer cell growth, while T beta R-III may function in the extracellular matrix.

  20. Electrochemical bioplatforms for the simultaneous determination of interleukin (IL)-8 mRNA and IL-8 protein oral cancer biomarkers in raw saliva.

    Science.gov (United States)

    Torrente-Rodríguez, R M; Campuzano, S; Ruiz-Valdepeñas Montiel, V; Gamella, M; Pingarrón, J M

    2016-03-15

    The development of electrochemical magnetobiosensors for the simultaneous determination of two biomarkers associated with salivary oral cancer, protein IL-8 and its messenger RNA (IL-8 mRNA) associated, in undiluted human saliva samples is reported in this work. The implemented methodology involves the use of functionalized magnetic beads, specific antibodies against IL-8 protein, a specific hairpin DNA sequence for IL-8 mRNA and amperometric detection at disposable dual screen printed carbon electrodes. This methodology exhibits high sensitivity and selectivity for the target analytes providing detection limits of 0.21 nM for IL-8 mRNA and 72.4 pgmL(-1) (far below the clinical established cut-off of 600 pgmL(-1)) for IL-8 protein in undiluted saliva samples. The dual amperometric magnetobiosensor was applied to the direct determination of both biomarkers in spiked raw saliva samples and to determine the endogenous content of IL-8 protein in saliva samples from 7 healthy individuals. The obtained results were statistically in agreement with those provided by a commercial ELISA kit.

  1. Transition from embryonic to adult epidermis in reptiles occurs by the production of corneous beta-proteins.

    Science.gov (United States)

    Alibardi, Lorenzo

    2014-01-01

    The adaptation of the epidermis in amniote vertebrates to life on land took place by a drastic change from an embryonic epidermis made of two-four periderm layers to a terrestrial-proof epidermis. This transition occurred by the increase in types and number of specialized corneous proteins coded by genes of the Epidermal Differentiation Complex. The prevalent types of corneous proteins produced in the reptilian epidermis contain a beta-sheet region of high amino acid homology which allows their polymerization into a meshwork of filaments forming the hard corneous material of scales and claws. The present immunogold ultrastructural study shows that this transition occurs with the synthesis of glycine-rich corneous beta-proteins (formerly indicated as beta-keratins) that are added to the initial framework of acidic intermediate filaments produced in the embryonic epidermis of lizards, snake, alligator and turtle. These corneous beta-proteins are accumulated in the transitional and definitive layers of reptilian epidermis formed underneath the transitory two-four layered embryonic epidermis. In the more specialized reptiles capable of shedding the epidermis as a single unit, such as lizards and snakes, special glycine-cysteine rich beta-proteins are initially produced in a single layer immediately formed beneath the embryonic epidermis, the oberhautchen. The latter layer allows the in ovo shedding of the embryonic epidermis in preparation for hatching, and in the following shedding cycles of the adult epidermis. The production of specialized corneous-specific beta-proteins in addition to intermediate filament keratins was probably an essential addition for terrestrial life during the evolution of reptiles into different lineages, including birds. The increase of glycine and cysteine in epidermal proteins enhanced the hydrophobicity, insolubility and mechanical strength of the stratum corneum in these amniotes.

  2. The experimental study of the effects of Radix Rehmanniae on the expression of the forth lumbar vertebrae osseous integrin beta 1 (Itg β1)mRNA in ovariectomized osteoporosis model rats%地黄对去卵巢骨质疏松大鼠腰椎骨组织整合素β1 mRNA 表达的影响

    Institute of Scientific and Technical Information of China (English)

    李小林; 韩红伟; 李彬; 武密山; 朱紫薇; 邓勇存; 叶圆圆; 赵素芝; 任立中; 王茹; 白霞

    2015-01-01

    目的:探讨地黄对去卵巢骨质疏松大鼠股骨骨密度、腰椎骨组织整合素β1 mRNA 表达的影响。方法72只雌性大鼠,随机分为假手术组、模型组、雌二醇组和地黄小、中、大剂量组。假手术组仅行假手术,其余五组行卵巢切除术。术后1 w 分别灌胃给予17β-雌二醇和地黄小、中、大剂量,连续给药3个月。测定血钙、磷和碱性磷酸酶( ALP),尿钙( u-Ca)、磷、尿脱氧吡啶酚( D-Pyr)和肌酐( Cr)。之后处死动物,取出右侧股骨,测定骨密度;取出第2腰椎,测定骨钙( b-Ca)、磷( b-P)含量;取出第4腰椎,采用实时荧光逆转录聚合酶链反应测定腰椎骨整合素β1 mRNA 表达。结果与假手术组相比,模型组血清 ALP、u-Ca、D-Pyr/Cr 显著增加,股骨密度、腰椎骨整合素β1 mRNA 表达均降低。与模型组比较,地黄中、大剂量组和雌二醇组均可使血清 ALP、u-Ca、D-Pyr/Cr 排出量降低,股骨密度、腰椎骨整合素β1 mRNA 表达均增加。结论地黄能抑制由于去卵巢雌激素缺乏引发的骨转换增强,提高骨密度,促进腰椎骨组织整合素β1 mRNA 表达,增强骨质量。%Objective To explore the influence of Radix Rehmanniae on bone mineral density ( BMD) of femur,the expression of the forth lumbar vertebrae osseous integrin beta 1(Itgβ1)mRNA in ovariectomized osteoporosis model rats .Mtehods 72 Sprague Dawley fe-male rats were randomly divided into sham ,model,17 β-estrogen treated and Radix Rehmanniae treated groups (12.5, 2.5 , 5 ml/kg).The sham group was only sham operated and the other five groups were ovariectomized respectively .One week after ovariectomy ,the rats were giv-en Radix Rehmanniae(1.25, 2.5, 5 ml/kg) and 17 β-estrogen(2 mg/kg)by intragastric administration for three months sustainedly .Serum contents of calcium(s-Ca),phosphorus(s-P),alkaline phosphate(ALP)and levels of urinary deoxypyridinoline

  3. Identification of the RNA recognition element of the RBPMS family of RNA-binding proteins and their transcriptome-wide mRNA targets.

    Science.gov (United States)

    Farazi, Thalia A; Leonhardt, Carl S; Mukherjee, Neelanjan; Mihailovic, Aleksandra; Li, Song; Max, Klaas E A; Meyer, Cindy; Yamaji, Masashi; Cekan, Pavol; Jacobs, Nicholas C; Gerstberger, Stefanie; Bognanni, Claudia; Larsson, Erik; Ohler, Uwe; Tuschl, Thomas

    2014-07-01

    Recent studies implicated the RNA-binding protein with multiple splicing (RBPMS) family of proteins in oocyte, retinal ganglion cell, heart, and gastrointestinal smooth muscle development. These RNA-binding proteins contain a single RNA recognition motif (RRM), and their targets and molecular function have not yet been identified. We defined transcriptome-wide RNA targets using photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) in HEK293 cells, revealing exonic mature and intronic pre-mRNA binding sites, in agreement with the nuclear and cytoplasmic localization of the proteins. Computational and biochemical approaches defined the RNA recognition element (RRE) as a tandem CAC trinucleotide motif separated by a variable spacer region. Similar to other mRNA-binding proteins, RBPMS family of proteins relocalized to cytoplasmic stress granules under oxidative stress conditions suggestive of a support function for mRNA localization in large and/or multinucleated cells where it is preferentially expressed.

  4. Protein phosphatase magnesium-dependent 1δ (PPM1D mRNA expression is a prognosis marker for hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Guang-Bing Li

    Full Text Available BACKGROUND: Protein phosphatase magnesium-dependent 1δ (PPM1D is an oncogene, overexpressed in many solid tumors, including ovarian cancer and breast cancer. The current study examined the expression and the prognostic value of PPM1D mRNA in human hepatocellular carcinoma (HCC. METHODS: Total RNA was extracted from 86 HCC and paired non-cancerous liver tissues. PPM1D mRNA expression was determined by real-time quantitative reverse transcriptase-polymerase chain reaction (qPCR. Immunohistochemistry assay was used to verify the expression of ppm1d protein in the HCC and non-cancerous liver tissues. HCC patients were grouped according to PPM1D mRNA expression with the average PPM1D mRNA level in non-cancerous liver tissue samples as the cut-off. Correlations between clinicopathologic variables, overall survival and PPM1D mRNA expression were analyzed. FINDINGS: PPM1D mRNA was significantly higher in HCC than in the paired non-cancerous tissue (p<0.01. This was confirmed by ppm1d staining. 56 patients were classified as high expression group and the other 30 patients were categorized as low expression group. There were significant differences between the two groups in term of alpha-fetoprotein (α-FP level (p<0.01, tumor size (p<0.01, TNM stage (p<0.01, recurrence incidence (p<0.01 and family history of liver cancer (p<0.01. The current study failed to find significant differences between the two groups in the following clinical characteristics: age, gender, portal vein invasion, lymphnode metastasis, hepatitis B virus (HBV infection and alcohol intake. Survival time of high expression group was significantly shorter than that of low expression group (median survival, 13 months and 32 months, respectively, p<0.01. CONCLUSION: Up-regulation of PPM1D mRNA was associated with progressive pathological feature and poor prognosis in HCC patients. PPM1D mRNA may serve as a prognostic marker in HCC.

  5. Intestinal cellular localization of PCNA protein and CYP1A mRNA in Atlantic salmon Salmo salar L. exposed to a model toxicant

    Directory of Open Access Journals (Sweden)

    Olsvik Pål A

    2009-03-01

    Full Text Available Abstract Background The aim of the study was to examine the intestinal cellular localization of proliferating cell nuclear antigen (PCNA and cytochrome P450 A1 (CYP1A expression in Atlantic salmon Salmo salar L. exposed to a model toxicant. The stress response was induced by intraperitoneal injection of four salmon with a single dose (50 mg/kg of the CYP1A inducer β-naphthoflavone (BNF and intestinal tissue (mid and distal intestine; MI and DI was sampled seven days later. Samples for histology and gene transcription analysis were collected from four exposed fish and four control fish. PCNA was assessed by immunohistochemistry, CYP1A mRNA was studied by in situ hybridization (ISH and finally the transcription of five genes was quantified by real-time quantitative RT-PCR (real-time RT-PCR; two detoxifying genes (CYP1A and glutathione S-transferase; GST, a stress marker gene (heat shock protein 70; HSP70, PCNA and a gene marker of apoptosis (caspase 6A. Results PCNA protein and CYP1A mRNA were successfully localized in the intestinal cells (MI of both experimental groups. At the cellular level, BNF significantly lowered intestinal cell proliferation and increased the CYP1A mRNA levels compared to the control group. The real-time RT-PCR data, which showed an increased mRNA expression both in the MI and DI of 139- and 62-fold, respectively, confirmed the increased cellular CYP1A mRNA levels detected using ISH. HSP70 expression was also up-regulated in the exposed fish. The other examined genes did not show any differential regulation in the experimental fish group. Conclusion This study showed that CYP1A mRNA had a specific intestinal cellular transcription pattern in Atlantic salmon exposed to BNF. At the cellular level CYP1A mRNA expression was always observed at or around the cell nucleus close to the basolateral cell membrane and at the tissue level CYP1A mRNA expression was most frequently observed in the basal and apex area of the intestinal

  6. beta-Tryptase up-regulates vascular endothelial growth factor expression via proteinase-activated receptor-2 and mitogen-activated protein kinase pathways in bone marrow stromal cells in acute myeloid leukemia.

    Science.gov (United States)

    Yang, Xiu-Peng; Li, Yan; Wang, Yazhu; Wang, Yue; Wang, Pingping

    2010-08-01

    Tryptases are predominantly mast cell-specific serine proteases with pleiotropic biological activities. Recently, significant amounts of tryptases have been shown to be produced by myeloblasts in certain patients with acute myeloid leukemia (AML), but the function of secreted tryptases in pathological circumstances remains unknown. In this study, we investigated whether beta-tryptase affects the expression of vascular endothelial growth factor (VEGF) in bone marrow stromal cells (BMSCs) in AML. We detected the expression of proteinase-activated receptor-2 (PAR-2) on AML BMSCs and found that beta-tryptase significantly up-regulated VEGF mRNA and protein expression in a dose-dependent manner by real-time PCR, Western blot, and ELISA. Furthermore, beta-tryptase increased ERK1/2 and p38MAPK phosphorylation, and pretreatment with FLLSY-NH(2), PD98059, and SB230580 (PAR-2, ERK1/2, and p38MAPK inhibitors, respectively) inhibited the beta-tryptase-induced production of VEGF. These results suggest that beta-tryptase up-regulates VEGF production in AML BMSCs via the PAR-2, ERK1/2, and p38MAPK signaling pathways.

  7. LPS-induced inflammation in the chicken is associated with CCAAT/enhancer binding protein beta-mediated fat mass and obesity associated gene down-regulation in the liver but not hypothalamus.

    Science.gov (United States)

    Zhang, Yanhong; Guo, Feng; Ni, Yingdong; Zhao, Ruqian

    2013-12-17

    The fat mass and obesity associated gene (FTO) is widely investigated in humans regarding its important roles in obesity and type 2 diabetes. Studies in mammals demonstrate that FTO is also associated with inflammation markers. However, the association of FTO with inflammation in chickens remains unclear. In this study, male chickens on day 28 posthatching were injected intraperitoneally with lipopolysaccharide (LPS) or saline to investigate whether the FTO gene is involved in LPS-induced inflammation. We detected significant down-regulation of FTO mRNA in the liver (P hypothalamus, 2 and 24 h after LPS challenge. Toll-like receptor (TLR) 2 (P hypothalamus. IL-1β was dramatically up-regulated (P hypothalamus 2 h after LPS challenge, while activation of IL-6 was observed in the liver (P hypothalamus. The 5'-flanking sequence of the chicken FTO gene contains nine predicted binding sites for CCAAT/enhancer binding protein beta (C/EBP beta) and one for signal transducer and activator of transcription 3 (STAT3). Significant elevation of C/EBP beta was detected in the liver (P hypothalamus, 2 h after LPS challenge. Lipopolysaccharide challenge increased the C/EBP beta binding to FTO promoter in the liver (P hypothalamus, is affected by the i.p. injection of LPS, which may be mediated through tissue-specific FTO transcriptional regulation by C/EBP beta and STAT3 interaction.

  8. High lib mRNA expression in breast carcinomas.

    Science.gov (United States)

    Satoh, Kazuki; Hata, Mitsumi; Yokota, Hiroshi

    2004-06-30

    Lib, first identified as a novel beta-amyloid responsive gene in rat astrocytes, has an extracellular domain of 15 leucine-rich repeats (LRRs) followed by a transmembrane domain and a short cytoplasmic region. It is a distinctly inducible gene and is thought to play a key role in inflammatory states via the LRR extracellular motif, an ideal structural framework for protein-protein and protein-matrix interactions. To evaluate potential roles of Lib, we screened various tumors for Lib expression. Lib mRNA expression was high and uniquely expressed in breast tumor tissues, compared to paired normal breast tissues. Lib mRNA was localized in the ductal carcinoma cells and Lib protein displayed a homophilic association on the surface of cultured cells. These data suggest that Lib may play a role in the progression of breast carcinomas and may be a diagnostic marker for breast tumors.

  9. Nitrogen and Crude Proteins in Beetroot (Beta vulgaris var. conditiva under Different Fertilization Treatments

    Directory of Open Access Journals (Sweden)

    Marko PETEK

    2012-11-01

    Full Text Available The research aim was to determine the influence of different organic and mineral fertilization treatments and post-harvest treatments on the content of nitrogen and crude proteins in the edible part of beetroot (Beta vulgaris var. conditiva. A field trial (2003-2005 was set up in a hilly part of Croatia according to the Latin square method with four types of fertilization (control, 50 t ha-1 stable manure, 500 and 1000 kg ha-1 NPK 5-20-30, while treatments involved harvested fresh beetroot and stored fresh beetroot. The highest dry weight (DW content was determined in climatologically favourable 2004 (average 14.8% DW and in the treatment with 1000 kg ha-1 NPK 5-20-30 (15.6% DW in harvested beetroot. In 2004 and 2005, the highest levels of nitrogen and crude proteins in harvested beetroot were determined in the treatment with 1000 kg ha-1 NPK 5-20-30 (2.41 and 2.43 g N kg-1 in fresh weight and 15.07 and 15.21 g crude proteins kg-1 in fresh weight, respectively. Regardless of fertilization treatment or studied year, nitrogen and crude protein contents were higher in stored than in harvested beetroot, by 12% on average. The lowest crude protein content was determined in treatment with stable manure what confirmed that protein content decreased by organic fertilization. It can be concluded that beetroot lost some water during the storage period, which increased its content of nitrogen and crude proteins in fresh weight and thus increased the nutritional quality of beetroot as a functional food.

  10. Plasma pre beta-HDL formation is decreased by atorvastatin treatment in type 2 diabetes mellitus : Role of phospholipid transfer protein

    NARCIS (Netherlands)

    Dallinga-Thie, G. M.; van Tol, A.; Dullaart, R. P. F.

    2009-01-01

    Atorvastatin lowers plasma phospholipid transfer protein (PLTP) activity, which stimulates pre-beta-HDL, generation in vitro. We determined the effect of atorvastatin on pre-beta-HDL formation and its relation with PLTP activity in type 2 diabetes. Methods: Plasma pre-beta-HDL formation as well as p

  11. Interconverting conformations of variants of the human amyloidogenic protein beta2-microglobulin quantitatively characterized by dynamic capillary electrophoresis and computer simulation

    DEFF Research Database (Denmark)

    Heegaard, Niels H H; Jørgensen, Thomas J D; Cheng, Lei

    2006-01-01

    Capillary electrophoretic separation profiles of cleaved variants of beta2-microglobulin (beta2m) reflect the conformational equilibria existing in solutions of these proteins. The characterization of these equilibria is of interest since beta2m is responsible for amyloid formation in dialysis-re...

  12. Exercise induced regulation of muscular Na+,K+ pump, FXYD1, and NHE1 mRNA and protein expression: importance of training status, intensity, and muscle type

    DEFF Research Database (Denmark)

    Rasmussen, Martin Krøyer; Juel, Carsten; Nordsborg, Nikolai Baastrup

    2011-01-01

    It is investigated if exercise induced mRNA changes cause similar protein expression changes of Na(+), K(+) pump isoforms (a1, a2, ß1, ß2), FXYD1 and NHE1 in rat skeletal muscle. Expression was evaluated (n=8 per group) in Soleus and EDL after 1 day, 3 days and 3 weeks (5 sessions per week......) of either sprint (4 x 3 min sprint + 1 min rest) or endurance (20 min) running. Two hours after exercise on day 1, no change in protein expression was apparent in either training group or muscle, whereas sprint exercise increased the mRNA of Soleus a2 (4.9±0.8 fold; P...

  13. Compound Danshen tablets downregulate amyloid protein precursor mRNA expression in a transgenic cell model of Alzheimer's disease Effects and a comparison with donepezil

    Institute of Scientific and Technical Information of China (English)

    Ren'an Qin; Desheng Zhou; Jiajun Wang; Hua Hu; Yang Yang; Xiaoxuan Yao; Xiaopeng Sun

    2012-01-01

    After gene mutation, the pcDNA3.1/APP595/596 plasmid was transfected into HEK293 cells to establish a cell model of Alzheimer’s disease. The cell model was treated with donepezil or compound Danshen tablets after culture for 72 hours. Reverse transcription-PCR showed that the mRNA expression of amyloid protein precursor decreased in all groups following culture for 24 hours, and that there was no significant difference in the amount of decrease between donepezil and compound Danshen tablets. Our results suggest that compound Danshen tablets can reduce expression of the mRNA for amyloid protein precursor in a transgenic cell model of Alzheimer’s disease, with similar effects to donepezil.

  14. The Escherichia coli antiterminator protein BglG stabilizes the 5' region of the bgl mRNA

    Indian Academy of Sciences (India)

    Abhilasha Gulati; S Mahadevan

    2001-06-01

    The -glucoside utilization (bgl) genes of Escherichia coli are positively regulated by the product of the bglG gene, which functions as an antiterminator by binding to specific sequences present within the bgl mRNA. BglG is inactivated by phosphorylation in the absence of -glucosides by BglF, the bgl-specific component of the phosphotransferase system (PTS). Here, we present evidence for an additional function for BglG, namely the stabilization of the 5— end of the bgl mRNA. Half-life measurements of the promoter-proximal region of the bgl mRNA indicate a five fold enhancement of stability in the presence of active (unphosphorylated) BglG. This enhancement is lost when the binding of BglG to mRNA is prevented by deletion of the binding site. Interestingly, stabilization by BglG does not extend to downstream sequences. The enhanced stability of the upstream sequences suggest that BglG remains bound to its target on the mRNA even after the downstream sequences have been degraded. Implications of these observations for the mechanism of positive regulation of the operon by BglG are discussed.

  15. Capping protein beta is required for actin cytoskeleton organisation and cell migration during Drosophila oogenesis.

    Science.gov (United States)

    Ogienko, Anna A; Karagodin, Dmitry A; Lashina, Valentina V; Baiborodin, Sergey I; Omelina, Eugeniya S; Baricheva, Elina M

    2013-02-01

    Capping protein (CP) is a well-characterised actin-binding protein important for regulation of actin filament (AF) assembly. CP caps the barbed end of AFs, inhibiting the addition and loss of actin monomers. In Drosophila melanogaster, the gene encoding CP β-subunit is named capping protein beta (cpb; see Hopmann et al. [1996] J Cell Biol 133: 1293-305). The cpb level is reduced in the Drosophila bristle actin cytoskeleton and becomes disorganised with abnormal morphology. A reduced level of the CP protein in ovary results in disruption of oocyte determination, and disturbance of nurse cell (NC) cortical integrity and dumping. We describe novel defects appearing in cpb mutants during oogenesis, in which cpb plays an important role in border and centripetal follicle cell migration, ring canal development and cytoplasmic AF formation. The number of long cytoplasmic AFs was dramatically reduced in cpb hypomorphs and abnormal actin aggregates was seen on the inner side of NC membranes. A hypothesis to explain the formation of abnormal short-cut cytoplasmic AFs and actin aggregates in the cpb mutant NCs was proffered, along with a discussion of the reasons for 'dumpless' phenotype formation in the mutants.

  16. MD-simulations of Beta-Amyloid Protein Insertion Efficiency and Kinetics into Neuronal Membrane Mimics

    Science.gov (United States)

    Qiu, Liming; Buie, Creighton; Vaughn, Mark; Cheng, Kwan

    2011-03-01

    Early interaction events of beta-amyloid (A β) peptides with the neuronal membranes play a key role in the pathogenesis of Alzheimer's disease. We have used all-atom MD simulations to study the protein insertion efficiency and kinetics of monomeric A β40 and A β42 into phosphatidylcholine lipid bilayers (PC) with and without 40 mole% cholesterol (CHOL) that mimic the cholesterol-enriched and depleted lipid nanodomains of the neuronal plasma membranes. Independent replicates of 200-ns simulations of each protein pre-inserted in the upper lipid layer were generated. In PC bilayers, only 25% of A β40 and 50% of A β42 in the replicates showed complete insertion into the lower lipid layer, whereas the percentages increased to 50% and 100%, respectively, in PC/CHOL bilayers, providing evidence that cholesterol improves the protein insertion efficiency into the bilayers. The rate of protein insertion was proportional to the hydrophobic, transmembrane helix length of the inserted peptide and depended on the cholesterol content. We propose that the lysine snorkeling and C-terminus anchoring of A β to the PC headgroups at the upper and lower lipid/water interfaces represent the dual-transmembrane stabilization mechanisms of A β in the neuronal membrane domains.

  17. Amyloid beta modulated the selectivity of heme-catalyzed protein tyrosine nitration: an alternative mechanism for selective protein nitration.

    Science.gov (United States)

    Yuan, Can; Li, Hailing; Gao, Zhonghong

    2012-10-01

    Protein tyrosine nitration is a post-translational modification associated with numerous pathological conditions. The biological consequences of this modification strongly depend on the site selectivity. Unfortunately, to date there is still no reliable model for predicting the selectivity of protein tyrosine nitration. Previously, we found that amyloid beta (Aβ) changed the selectivity of enolase tyrosine nitration upon binding to heme. It seemed that there was a link between the hydrophilicity of Aβ and the site-specific tyrosine nitration. We further investigated the role of the hydrophilicity of the molecules that bind to heme in the selectivity of protein tyrosine nitration. We found that Aβ(1-16), Aβ(1-20), and Aβ(1-40), upon binding to heme and interacting with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in a site-specific manner, differently modulated the site selectivity of heme-catalyzed GAPDH tyrosine nitration. The modulation is associated with the hydrophilicity of the Aβ peptides, which changed the surrounding environment of the heme. At the same time, the Aβ-heme complexes were found to be more effective at inactivating GAPDH than heme alone, and the selective tyrosine nitration that was catalyzed by Aβ-heme played an important role. These findings suggest an alternative mechanism for the selectivity of protein tyrosine nitration, which may lead to a better understanding of the factors that influence protein tyrosine nitration selectivity and the important roles of Aβ and heme in the pathogenesis of Alzheimer's disease, where Aβ accumulation and Aβ-dependent protein nitration play central roles.

  18. Structural similarity of a developmentally regulated bacterial spore coat protein to beta gamma-crystallins of the vertebrate eye lens.

    Science.gov (United States)

    Bagby, S; Harvey, T S; Eagle, S G; Inouye, S; Ikura, M

    1994-05-10

    The solution structure of Ca(2+)-loaded protein S (M(r) 18,792) from the Gram-negative soil bacterium Myxococcus xanthus has been determined by multidimensional heteronuclear NMR spectroscopy. Protein S consists of four internally homologous motifs, arranged to produce two domains with a pseudo-twofold symmetry axis, overall resembling a triangular prism. Each domain consists of two topologically inequivalent "Greek keys": the second and fourth motifs form standard Greek keys, whereas the first and third motifs each contain a regular alpha-helix in addition to the usual four beta-strands. The structure of protein S is similar to those of the vertebrate eye lens beta gamma-crystallins, which are thought to be evolutionarily related to protein S. Both protein S and the beta gamma-crystallins function by forming stable multimolecular assemblies. However, protein S possesses distinctive motif organization and domain packing, indicating a different mode of oligomerization and a divergent evolutionary pathway from t