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  1. Irbesartan inhibits advanced glycation end product (AGE)-induced up-regulation of vascular cell adhesion molecule-1 (VCAM-1) mRNA levels in glomerular endothelial cells.

    Science.gov (United States)

    Matsui, Takanori; Nishino, Yuri; Maeda, Sayaka; Takeuchi, Masayoshi; Yamagishi, Sho-ichi

    2011-05-01

    Renin-angiotensin system (RAS) plays a central role in the development and progression of diabetic nephropathy. There is a growing body of evidence that advanced glycation end products (AGE) and inflammation contribute to diabetic nephropathy as well. However, the pathophysiological crosstalk between the RAS and AGE in inflammatory reactions in glomerular endothelial cells (ECs) remains unknown. In this study, we examined whether and how irbesartan, an angiotensin II type 1 receptor blocker (ARB), inhibited the AGE-induced vascular cell adhesion molecule-1 (VCAM-1) gene expression in cultured human glomerular ECs. Irbesartan or an anti-oxidant N-acetylcysteine inhibited the AGE-induced increase in reactive oxygen species (ROS) generation and subsequently blocked up-regulation of VCAM-1 mRNA levels in glomerular ECs. AGE significantly stimulated angiotensin II production by glomerular ECs. Furthermore, irbesartan completely suppressed up-regulation of VCAM-1 mRNA levels in AGE plus angiotensin II-exposed glomerular ECs. Our present data suggest that there exists a crosstalk between the RAS and AGE in inflammatory reactions in glomerular ECs. Irbesartan may play a protective role against diabetic nephropathy by blocking the deleterious effects of AGE-elicited angiotensin II and ROS. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Stress granules: sites of mRNA triage that regulate mRNA stability and translatability.

    Science.gov (United States)

    Kedersha, N; Anderson, P

    2002-11-01

    Mammalian stress granules (SGs) are cytoplasmic domains into which mRNAs are sorted dynamically in response to phosphorylation of eukaryotic initiation factor (eIF) 2alpha, a key regulatory step in translational initiation. The activation of one or more of the eIF2alpha kinases leads to SG assembly by decreasing the levels of eIF2-GTP-tRNA(Met), the ternary complex that is normally required for loading the initiator methionine onto the 48 S preinitiation complex to begin translation. This stress-induced scarcity of eIF2-GTP-tRNA(Met) allows the RNA-binding proteins TIA-1 (T-cell internal antigen-1) and TIAR (TIA-1-related protein) to bind the 48 S complex in lieu of the ternary complex, thereby promoting polysome disassembly and the concurrent routing of the mRNA into a SG. The actual formation of SGs occurs upon auto-aggregation of the prion-like C-termini of TIA-1 proteins; this aggregation is reversed in vivo by overexpression of the heat-shock protein (HSP) chaperone HSP70. Remarkably, HSP70 mRNA is excluded from SGs and is preferentially translated during stress, indicating that the RNA composition of the SG is selective. Moreover, the effects of HSP70 on TIA aggregation suggest a feedback loop whereby HSP70 synthesis is auto-regulated. Proteins that promote mRNA stability [e.g. HuR (Hu protein R)] and destabilize mRNA [i.e. tristetraprolin (TTP)] are also recruited to SGs, suggesting that SGs effect a process of mRNA triage, by promoting polysome disassembly and routing mRNAs to cytoplasmic domains enriched for HuR and TTP. This model reveals connections between the eIF2alpha kinase system, mRNA stability and cellular chaperone levels.

  3. Cup regulates oskar mRNA stability during oogenesis.

    Science.gov (United States)

    Broyer, Risa M; Monfort, Elena; Wilhelm, James E

    2017-01-01

    The proper regulation of the localization, translation, and stability of maternally deposited transcripts is essential for embryonic development in many organisms. These different forms of regulation are mediated by the various protein subunits of the ribonucleoprotein (RNP) complexes that assemble on maternal mRNAs. However, while many of the subunits that regulate the localization and translation of maternal transcripts have been identified, relatively little is known about how maternal mRNAs are stockpiled and stored in a stable form to support early development. One of the best characterized regulators of maternal transcripts is Cup - a broadly conserved component of the maternal RNP complex that in Drosophila acts as a translational repressor of the localized message oskar. In this study, we have found that loss of cup disrupts the localization of both the oskar mRNA and its associated proteins to the posterior pole of the developing oocyte. This defect is not due to a failure to specify the oocyte or to disruption of RNP transport. Rather, the localization defects are due to a drop in oskar mRNA levels in cup mutant egg chambers. Thus, in addition to its role in regulating oskar mRNA translation, Cup also plays a critical role in controlling the stability of the oskar transcript. This suggests that Cup is ideally positioned to coordinate the translational control function of the maternal RNP complex with its role in storing maternal transcripts in a stable form. Published by Elsevier Inc.

  4. Post-transcriptional gene regulation by mRNA modifications

    Science.gov (United States)

    Zhao, Boxuan Simen; Roundtree, Ian A.; He, Chuan

    2016-01-01

    The recent discovery of reversible mRNA methylation has opened a new realm of post-transcriptional gene regulation in eukaryotes. The identification and functional characterization of proteins that specifically recognize RNA N6-methyladenosine (m6A) unveiled it as a modification that cells utilize to accelerate mRNA metabolism and translation. N6-adenosine methylation directs mRNAs to distinct fates by grouping them for differential processing, translation and decay in processes such as cell differentiation, embryonic development and stress responses. Other mRNA modifications, including N1-methyladenosine (m1A), 5-methylcytosine (m5C) and pseudouridine, together with m6A form the epitranscriptome and collectively code a new layer of information that controls protein synthesis. PMID:27808276

  5. Regulation of mRNA translation during mitosis.

    Science.gov (United States)

    Tanenbaum, Marvin E; Stern-Ginossar, Noam; Weissman, Jonathan S; Vale, Ronald D

    2015-08-25

    Passage through mitosis is driven by precisely-timed changes in transcriptional regulation and protein degradation. However, the importance of translational regulation during mitosis remains poorly understood. Here, using ribosome profiling, we find both a global translational repression and identified ~200 mRNAs that undergo specific translational regulation at mitotic entry. In contrast, few changes in mRNA abundance are observed, indicating that regulation of translation is the primary mechanism of modulating protein expression during mitosis. Interestingly, 91% of the mRNAs that undergo gene-specific regulation in mitosis are translationally repressed, rather than activated. One of the most pronounced translationally-repressed genes is Emi1, an inhibitor of the anaphase promoting complex (APC) which is degraded during mitosis. We show that full APC activation requires translational repression of Emi1 in addition to its degradation. These results identify gene-specific translational repression as a means of controlling the mitotic proteome, which may complement post-translational mechanisms for inactivating protein function.

  6. Regulation of endothelial cell nitric oxide synthase mRNA expression by shear stress.

    Science.gov (United States)

    Uematsu, M; Ohara, Y; Navas, J P; Nishida, K; Murphy, T J; Alexander, R W; Nerem, R M; Harrison, D G

    1995-12-01

    Shear stress enhances expression of Ca(2+)-calmodulin-sensitive endothelial cell nitric oxide synthase (ecNOS) mRNA and protein in bovine aortic endothelial cells (BAEC). The present studies were performed to investigate mechanisms responsible for regulation of ecNOS mRNA expression by shear stress and to determine if this induction of ecNOS mRNA is accompanied by an enhanced nitric oxide (NO) production. Shear stresses of 15 dyn/cm2 for 3-24 h resulted in a two- to threefold increase of ecNOS mRNA content quantified by Northern analysis in BAEC. Shear stresses (1.2-15 dyn/cm2) for 3 h resulted in an induction of ecNOS mRNA in a dose-dependent manner. In human aortic endothelial cells, shear stresses of 15 dyn/cm2 for 3 h also resulted in ecNOS mRNA induction. In BAEC, this induction in ecNOS mRNA was prevented by coincubation with actinomycin D (10 micrograms/ml). The K+ channel antagonist tetraethylammonium chloride (3 mM) prevented increase in ecNOS mRNA in response to shear stress. The ecNOS promotor contains putative binding domains for AP-1 complexes, potentially responsive to activation of protein kinase C (PKC). However, selective PKC inhibitor calphostin C (100 nM) did not inhibit ecNOS induction by shear stress. Finally, production of nitrogen oxides under both basal conditions and in response to the calcium ionophore A-23187 (1 microM) by BAEC exposed to shear stress was increased approximately twofold compared with cells not exposed to shear stress. These data suggest that ecNOS mRNA expression is regulated by K+ channel opening, but not by activation of PKC, and that shear not only enhances ecNOS mRNA expression but increases capacity of endothelial cells to release NO.

  7. The coupled and uncoupled mechanisms by which trans-acting factors regulate mRNA stability and translation.

    Science.gov (United States)

    Fukao, Akira; Fujiwara, Toshinobu

    2017-04-01

    In mammals, spatiotemporal control of protein synthesis plays a key role in the post-transcriptional regulation of gene expression during cell proliferation, development and differentiation and RNA-binding proteins (RBPs) and microRNAs (miRNAs) are required for this phenomenon. RBPs and miRNAs control the levels of mRNA protein products by regulating mRNA stability and translation. Recent studies have shown that RBPs and miRNAs simultaneously regulate mRNA stability and translation, and that the differential functions of RBPs and miRNAs are dependent on their interaction partners. Here, we summarize the coupled- and uncoupled mechanisms by which trans-acting factors regulate mRNA stability and translation. © The Authors 2016. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  8. Digoxin up-regulates multidrug resistance transporter (MDR1) mRNA and simultaneously down-regulates steroid xenobiotic receptor mRNA.

    Science.gov (United States)

    Takara, Kohji; Takagi, Kentaro; Tsujimoto, Masayuki; Ohnishi, Noriaki; Yokoyama, Teruyoshi

    2003-06-20

    A steroid xenobiotic receptor (SXR) is involved in the induction of MDR1/P-glycoprotein. MDR1 up-regulation by digoxin was previously demonstrated in human colon adenocarcinoma Caco-2 cells, but the participation of SXR remains unclear. Herein, the participation of SXR in MDR1 up-regulation was examined using reverse transcription-polymerase chain reaction in Caco-2 cells, and digoxin-tolerant cells (Caco/DX) as well as human colon carcinoma LS180 cells, which expressed SXR. MDR1 mRNA expression in Caco-2 or LS180 cells was increased by exposure to 1 microM digoxin for 24h, in a concentration-dependent manner, but SXR mRNA decreased concentration-dependently and was undetectable or significantly lower at 1 microM digoxin, indicating antithetical changes in MDR1 and SXR mRNA expression. Moreover, the MDR1 mRNA level was higher in Caco/DX cells than Caco-2 cells, whereas the SXR mRNA level was lower in Caco/DX cells. Consequently, digoxin was demonstrated to up-regulate MDR1 mRNA and simultaneously down-regulate SXR mRNA expression.

  9. Regulation and dysregulation of vitellogenin mRNA accumulation in daphnids (Daphnia magna)

    Energy Technology Data Exchange (ETDEWEB)

    Hannas, Bethany R.; Wang, Ying H.; Thomson, Susanne; Kwon, Gwijun; Li Hong [Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC 27695-7633 (United States); LeBlanc, Gerald A., E-mail: Gerald_LeBlanc@ncsu.edu [Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC 27695-7633 (United States)

    2011-01-25

    The induction of vitellogenin in oviparous vertebrates has become the gold standard biomarker of exposure to estrogenic chemicals in the environment. This biomarker of estrogen exposure also has been used in arthropods, however, little is known of the factors that regulate the expression of vitellogenin in these organisms. We investigated changes in accumulation of mRNA products of the vitellogenin gene Vtg2 in daphnids (Daphnia magna) exposed to a diverse array of chemicals. We further evaluated the involvement of hormonal factors in the regulation of vitellogenin expression that may be targets of xenobiotic chemicals. Expression of the Vtg2 gene was highly responsive to exposure to various chemicals with an expression range spanning approximately four orders of magnitude. Chemicals causing the greatest induction were piperonyl butoxide, chlordane, 4-nonylphenol, cadmium, and chloroform. Among these, only 4-nonylphenol is recognized to be estrogenic. Exposure to several chemicals also suppressed Vtg2 mRNA levels, as much as 100-fold. Suppressive chemicals included cyproterone acetate, acetone, triclosan, and atrazine. Exposure to the estrogens diethylstilbestrol and bisphenol A had little effect on vitellogenin mRNA levels further substantiating that these genes are not induced by estrogen exposure. Exposure to the potent ecdysteroids 20-hydroxyecdysone and ponasterone A revealed that Vtg2 was subject to strong suppressive control by these hormones. Vtg2 mRNA levels were not significantly affected from exposure to several juvenoid hormones. Results indicate that ecdysteroids are suppressors of vitellogenin gene expression and that vitellogenin mRNA levels can be elevated or suppressed in daphnids by xenobiotics that elicit antiecdysteroidal or ecdysteroidal activity, respectively. Importantly, daphnid Vtg2 is not elevated in response to estrogenic activity.

  10. Regulation of mRNA Trafficking by Nuclear Pore Complexes

    Directory of Open Access Journals (Sweden)

    Amandine Bonnet

    2014-09-01

    Full Text Available Over the last two decades, multiple studies have explored the mechanisms governing mRNA export out of the nucleus, a crucial step in eukaryotic gene expression. During transcription and processing, mRNAs are assembled into messenger ribonucleoparticles (mRNPs. mRNPs are then exported through nuclear pore complexes (NPCs, which are large multiprotein assemblies made of several copies of a limited number of nucleoporins. A considerable effort has been put into the dissection of mRNA export through NPCs at both cellular and molecular levels, revealing the conserved contributions of a subset of nucleoporins in this process, from yeast to vertebrates. Several reports have also demonstrated the ability of NPCs to sort out properly-processed mRNPs for entry into the nuclear export pathway. Importantly, changes in mRNA export have been associated with post-translational modifications of nucleoporins or changes in NPC composition, depending on cell cycle progression, development or exposure to stress. How NPC modifications also impact on cellular mRNA export in disease situations, notably upon viral infection, is discussed.

  11. LARP6 Meets Collagen mRNA: Specific Regulation of Type I Collagen Expression

    Directory of Open Access Journals (Sweden)

    Yujie Zhang

    2016-03-01

    Full Text Available Type I collagen is the most abundant structural protein in all vertebrates, but its constitutive rate of synthesis is low due to long half-life of the protein (60–70 days. However, several hundred fold increased production of type I collagen is often seen in reparative or reactive fibrosis. The mechanism which is responsible for this dramatic upregulation is complex, including multiple levels of regulation. However, posttranscriptional regulation evidently plays a predominant role. Posttranscriptional regulation comprises processing, transport, stabilization and translation of mRNAs and is executed by RNA binding proteins. There are about 800 RNA binding proteins, but only one, La ribonucleoprotein domain family member 6 (LARP6, is specifically involved in type I collagen regulation. In the 5′untranslated region (5’UTR of mRNAs encoding for type I and type III collagens there is an evolutionally conserved stem-loop (SL structure; this structure is not found in any other mRNA, including any other collagen mRNA. LARP6 binds to the 5′SL in sequence specific manner to regulate stability of collagen mRNAs and their translatability. Here, we will review current understanding of how is LARP6 involved in posttranscriptional regulation of collagen mRNAs. We will also discuss how other proteins recruited by LARP6, including nonmuscle myosin, vimentin, serine threonine kinase receptor associated protein (STRAP, 25 kD FK506 binding protein (FKBP25 and RNA helicase A (RHA, contribute to this process.

  12. Coordinated Regulations of mRNA Synthesis and Decay during Cold Acclimation in Arabidopsis Cells.

    KAUST Repository

    Arae, Toshihiro

    2017-04-18

    Plants possess a cold acclimation system to acquire freezing tolerance through pre-exposure to non-freezing low temperatures. The transcriptional cascade of C-repeat binding factors (CBFs)/dehydration response element-binding factors (DREBs) is considered a major transcriptional regulatory pathway during cold acclimation. However, little is known regarding the functional significance of mRNA stability regulation in the response of gene expression to cold stress. The actual level of individual mRNAs is determined by a balance between mRNA synthesis and degradation. Therefore, it is important to assess the regulatory steps to increase our understanding of gene regulation. Here, we analyzed temporal changes in mRNA amounts and half-lives in response to cold stress in Arabidopsis cell cultures based on genome-wide analysis. In this mRNA decay array method, mRNA half-life measurements and microarray analyses were combined. In addition, temporal changes in the integrated value of transcription rates were estimated from the above two parameters using a mathematical approach. Our results showed that several cold-responsive genes, including Cold-regulated 15a, were relatively destabilized, whereas the mRNA amounts were increased during cold treatment by accelerating the transcription rate to overcome the destabilization. Considering the kinetics of mRNA synthesis and degradation, this apparently contradictory result supports that mRNA destabilization is advantageous for the swift increase in CBF-responsive genes in response to cold stress.

  13. Positive regulation of corneal type V collagen mRNA: analysis by chicken-human heterokaryon formation.

    Science.gov (United States)

    Linsenmayer, T F; Igoe, F; Gibney, E; Gordon, M K; Birk, D E

    1996-10-10

    Our previous studies have suggested that type V collagen is at least one factor responsible for the characteristically small, uniform diameter of striated collagen fibrils of the corneal stroma. These fibrils, which are heterotypic combinations of collagen types I and V, contain four- to fivefold more type V collagen than those of tendon and sclera. The latter are much larger and more heterodisperse. This high content of type V collagen in cornea is reflected by an equally elevated content of alpha1(V) chain mRNA in corneal fibroblasts. Thus, the increased production of the molecule in cornea appears to be regulated at the level of transcription and/or mRNA stability. One possible explanation for this is that corneal fibroblasts contain positive regulatory factors that specifically upregulate transcription of the type V collagen genes and/or increase their mRNA stability. To test this possibility, we have produced transient heterokaryons by fusing chicken corneal fibroblasts with two human noncorneal cell lines selected as containing little if any alpha1(V) mRNA. If the chicken corneal cells contain positive regulators that can act across species, these regulators should result in increased levels of the human alpha1(V) transcript. The results were evaluated by reverse transcript-polymerase chain reaction employing a primer pair selected for its ability specifically to amplify part of the human alpha1(V) mRNA. In fusions between chicken corneal fibroblasts and the human cell lines, after a lag of 10-14 h the heterokaryon-containing cultures showed de novo appearance or upregulation of human alpha1(V) chain mRNA, compared with that of the parental cell lines. Cultures of the mixed cell types that had not been fused showed no such upregulation, so the effect was not mediated by diffusible substances acting between the cells. Chicken tendon fibroblasts, a low producer of type V collagen, when tested in the same assay, evoked no detectible increase in the human

  14. Testosterone Regulates NUCB2 mRNA Expression in Male Mouse Hypothalamus and Pituitary Gland.

    Science.gov (United States)

    Seon, Sojeong; Jeon, Daun; Kim, Heejeong; Chung, Yiwa; Choi, Narae; Yang, Hyunwon

    2017-03-01

    Nesfatin-1/NUCB2 is known to take part in the control of the appetite and energy metabolism. Recently, many reports have shown nesfatin-1/NUCB2 expression and function in various organs. We previously demonstrated that nesfatin-1/NUCB2 expression level is higher in the pituitary gland compared to other organs and its expression is regulated by 17β-estradiol and progesterone secreted from the ovary. However, currently no data exist on the expression of nesfatin-1/NUCB2 and its regulation mechanism in the pituitary of male mouse. Therefore, we examined whether nesfatin-1/NUCB2 is expressed in the male mouse pituitary and if its expression is regulated by testosterone. As a result of PCR and western blotting, we found that a large amount of nesfatin-1/NUCB2 was expressed in the pituitary and hypothalamus. The NUCB2 mRNA expression level in the pituitary was decreased after castration, but not in the hypothalamus. In addition, its mRNA expression level in the pituitary was increased after testosterone treatment in the castrated mice, whereas, the expression level in the hypothalamus was significantly decreased after the treatment with testosterone. The in vitro experiment to elucidate the direct effect of testosterone on NUCB2 mRNA expression showed that NUCB2 mRNA expression was significantly decreased with testosterone in cultured hypothalamus tissue, but increased with testosterone in cultured pituitary gland. The present study demonstrated that nesfatin-1/NUCB2 was highly expressed in the male mouse pituitary and was regulated by testosterone. This data suggests that reproductive-endocrine regulation through hypothalamus-pituitary-testis axis may contribute to NUCB2 mRNA expression in the mouse hypothalamus and pituitary gland.

  15. FDA 101: Regulating Biological Products

    Science.gov (United States)

    ... Products For Consumers Home For Consumers Consumer Updates FDA 101: Regulating Biological Products Share Tweet Linkedin Pin ... and highly important field. What biological products does FDA regulate? The Center for Biologics Evaluation and Research ( ...

  16. Thyroid hormones regulate levels of thyrotropin-releasing-hormone mRNA in the paraventricular nucleus

    Energy Technology Data Exchange (ETDEWEB)

    Koller, K.J.; Wolff, R.S.; Warden, M.K.; Zoeller, R.T.

    1987-10-01

    Cellular levels of messenger RNA encoding thyrotropin-releasing hormone (TRH) were measured in the paraventricular nucleus of the hypothalamus and the reticular nucleus of the thalamus in male rats after chemical thyroidectomy and thyroid hormone, replacement. TRH mRNA levels were measured by quantitative in situ hybridization histochemistry using a /sup 35/S-labeled synthetic 48-base oligodeoxynucleotide probe and quantitative autoradiography. Chemical thyroidectomy, produced by the administration of 6-(n-propyl)-2-thiouracil (PrSur), reduced plasma thyroxine below detection limits and significantly increased TRH mRNA in the paraventricular nucleus. Treatments with exogenous L-triiodothyronine (T/sub 3/) reduced TRH mRNA to the same level in both hypothyroid and euthyroid animals. Neither PrSur treatment nor T/sub 3/ replacement influenced TRH mRNA levels in the reticular nucleus of the thalamus. Blot hybridization analysis of electrophoretically fractionated total RNA from pituitaries of these animals indicated that thyrotropin-..beta.. mRNA levels were elevated after thyroidectomy and reduced by T/sub 3/ treatment, showing that the pituitary-thyroid axis was indeed stimulated by PrSur treatment. These results suggest that thyroid hormones are involved, either directly or indirectly, in regulating the biosynthesis of TRH in the thyrotropic center of the hypothalamus.

  17. 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.

  18. 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.

  19. Regulation of endothelial cell nitric oxide synthase mRNA expression by shear stress

    National Research Council Canada - National Science Library

    Uematsu, M; Ohara, Y; Navas, J P; Nishida, K; Murphy, T J; Alexander, R W; Nerem, R M; Harrison, D G

    1995-01-01

    ... by an enhanced nitric oxide (NO) production. Shear stresses of 15 dyn/cm2 for 3-24 h resulted in a two- to threefold increase of ecNOS mRNA content quantified by Northern analysis in BAEC. Shear stresses (1.2-15 dyn/cm2...

  20. Vitamin D and the RNA transcriptome: more than mRNA regulation

    Directory of Open Access Journals (Sweden)

    Moray J Campbell

    2014-05-01

    Full Text Available The GRCh37.p13 primary assembly of the human genome contains 20805 protein coding mRNA, and 37147 non-protein coding genes and pseudogenes that as a result of RNA processing and editing generate 196501 gene transcripts. Given the size and diversity of the human transcriptome, it is timely to revisit what is known of VDR function in the regulation and targeting of transcription.Early transcriptomic studies using microarray approaches focused on the protein coding mRNA that were regulated by the VDR, usually following treatment with ligand. These studies quickly established the approxamte size, and surprising diversity of the VDR transcriptome, revealing it to be highly heterogenous and cell type and time dependent. With the discovery of microRNA, investigators also considered VDR regulation of these non-protein coding RNA. Again, cell and time dependency has emerged. Attempts to integrate mRNA and miRNA regulation patterns are beginning to reveal patterns of co-regulation and interaction that allow for greater control of mRNA expression, and the capacity to govern more complex cellular events. As the awareness of the diversity of non-coding RNA increases, it is evident that VDR actions are mediated through these molecules also. Key knowledge gaps remain over the VDR transcriptome. The causes for the cell and type dependent transcriptional heterogenetiy remain enigmatic. ChIP-Seq approaches have confirmed that VDR binding choices differ very significantly by cell type, but as yet the underlying causes distilling VDR binding choices are unclear. Similarly, it is clear that many of the VDR binding sites are non-canonical in nature but again the mechanisms underlying these interactions are unclear. Finally, although alternative splicing is clearly a very significant process in cellular transcriptional control, the lack of RNA-Seq data centered on VDR function are currently limiting the global assessment of the VDR transcriptome. VDR focused research

  1. Regulation and function of FTO mRNA expression in human skeletal muscle and subcutaneous adipose tissue

    DEFF Research Database (Denmark)

    Grunnet, Louise G; Nilsson, Emma; Ling, Charlotte

    2009-01-01

    Objective. Common variants in FTO (the fat-mass and obesity-associated gene) associate with obesity and type 2 diabetes. The regulation and biological function of FTO mRNA expression in target tissue is unknown. We investigated the genetic and non-genetic regulation of FTO mRNA in skeletal muscle......) and elderly (58-66 years) non-diabetic twins examined by a hyperinsulinemic euglycemic clamp including indirect calorimetry. FTO mRNA expression was determined in subcutaneous adipose tissue (n=226) and skeletal muscle biopsies (n=158). Results. Heritability of FTO expression in both tissues was low, and FTO...... expression was not influenced by FTO rs9939609 genotype. FTO mRNA expression in skeletal muscle was regulated by age and sex, whereas age and BMI were predictors of adipose tissue FTO mRNA expression. FTO mRNA expression in adipose tissue was associated with an atherogenic lipid profile. In skeletal muscle...

  2. Inflammation-regulated mRNA stability and the progression of vascular inflammatory diseases.

    Science.gov (United States)

    Herman, Allison B; Autieri, Michael V

    2017-11-15

    Cardiovascular disease remains a major medical and socioeconomic burden in developed and developing societies, and will increase with an aging and increasingly sedentary society. Vascular disease and atherosclerotic vascular syndromes are essentially inflammatory disorders, and transcriptional and post-transcriptional processes play essential roles in the ability of resident vascular and inflammatory cells to adapt to environmental stimuli. The regulation of mRNA translocation, stability, and translation are key processes of post-transcriptional regulation that permit these cells to rapidly respond to inflammatory stimuli. For the most part, these processes are controlled by elements in the 3'-UTR of labile, proinflammatory transcripts. Since proinflammatory transcripts almost exclusively contain AU-rich elements (AREs), this represents a tightly regulated and specific mechanism for initiation and maintenance of the proinflammatory phenotype. RNA-binding proteins (RBPs) recognize cis elements in 3'-UTR, and regulate each of these processes, but there is little literature exploring the concept that RBPs themselves can be directly regulated by inflammatory stimuli. Conceptually, inflammation-responsive RBPs represent an attractive target of rational therapies to combat vascular inflammatory syndromes. Herein we briefly describe the cellular and molecular etiology of atherosclerosis, and summarize our current understanding of RBPs and their specific roles in regulation of inflammatory mRNA stability. We also detail RBPs as targets of current anti-inflammatory modalities and how this may translate into better treatment for vascular inflammatory diseases. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  3. Differential regulation of renal cyclooxygenase mRNA by dietary salt intake

    DEFF Research Database (Denmark)

    Jensen, B L; Kurtz, A

    1997-01-01

    Experiments were done to investigate the influence of dietary salt intake on renal cyclooxygenase (COX) I and II mRNA levels. To this end rats were fed either a low NaCl diet (LS; 0.02% NaCl wt/wt) or a high NaCl diet (HS diet; 4% NaCl wt/wt) for 5, 10 and 20 days. After 10 days Na excretion...... differed 760-fold, plasma renin activity and renin mRNA were increased eight- and threefold in LS compared to HS animals. Total renal COX I mRNA decreased 50% following the LS diet and did not change after the HS diet. Conversely, COX II mRNA declined after HS intake and transiently increased after salt...... depletion. COX I and II mRNAs were unevenly distributed along the cortical-medullary axis with ratios of the cortex:outer medulla:papilla of 1:3:23 and 1:1:2, respectively. Cortical COX mRNAs were inversely regulated by salt intake with eightfold changes in COX II. Conversely, in medullary zones, COX I m...

  4. m6A RNA Modification Determines Cell Fate by Regulating mRNA Degradation.

    Science.gov (United States)

    Guo, Minjun; Liu, Xinhui; Zheng, Xiaotong; Huang, Yinghui; Chen, Xuechai

    2017-08-01

    Emerging evidence suggests that epitranscriptional modifications influence multiple cellular processes. N6-methyladenosine (m6A), as the most abundant reversible methylation of mRNA, has also been reported to play critical roles in modulating embryonic stem cell differentiation and somatic cell reprogramming by regulating gene expression. This review examined the characteristics of m6A, including the distribution profile and currently discovered "writer," "eraser," and "reader" proteins. Moreover, the hypothesis is proposed that m6A could influence cell fate determination, and the underlying mechanisms are due to the related mRNA degradation, causing weakening of previous cell characteristics and eventually leading them to develop into the reverse direction (pluripotency or differentiation state). Accordingly, m6A modifications presented its potential role in cell fate determination, which provides new insights into understanding the mechanisms of various diseases.

  5. Salinity Regulates Claudin mRNA and Protein Expression in the Teleost Gill

    DEFF Research Database (Denmark)

    Tipsmark, Christian K; Baltzegar, David A; Ozden, Ozkan

    2008-01-01

    The teleost gill carries out NaCl uptake in fresh water (FW) and NaCl excretion in seawater (SW). This transformation with salinity requires close regulation of ion transporter capacity and epithelial permeability. This study investigates the regulation of tight junctional claudins during salinity...... was localized deep in the FW gill filament, whereas staining was found apically in SW gill. Claudin 4-like proteins are localized predominantly in the filament outer epithelial layer and staining appears more intense in gill of FW versus SW fish. Additionally, tilapia claudin 28a and 30 genes were characterized......, and mRNA expression was found to increase during FW acclimation. These studies are the first to detect putative claudin proteins in teleosts and show their localization and regulation with salinity in gill epithelium. The data indicate that claudins may be important in permeability changes associated...

  6. Regulation of axon guidance by compartmentalized nonsense-mediated mRNA decay

    DEFF Research Database (Denmark)

    Colak, Dilek; Ji, Sheng-Jian; Porse, Bo T

    2013-01-01

    Growth cones enable axons to navigate toward their targets by responding to extracellular signaling molecules. Growth-cone responses are mediated in part by the local translation of axonal messenger RNAs (mRNAs). However, the mechanisms that regulate local translation are poorly understood. Here we...... (NMD) pathway. We find that NMD regulates Robo3.2 synthesis by inducing the degradation of Robo3.2 transcripts in axons that encounter the floor plate. Commissural neurons deficient in NMD proteins exhibit aberrant axonal trajectories after crossing the midline, consistent with misregulation of Robo3.......2 expression. These data show that local translation is regulated by mRNA stability and that NMD acts locally to influence axonal pathfinding....

  7. Involvement of the p53 and HPV-16 early 3'UTRs in mRNA regulation

    DEFF Research Database (Denmark)

    Glahder, Jacob-Andreas Harald

    Genregulering forekommer på mange niveauer og elementer både i og udenfor genets læseramme er vigtige. I denne afhandling analyseres elementer i den tidlige 3' ikke-translaterede region (3'UTR) af Human Papillomavirus type 16 (HPV-16) genomet og også af p53 3'UTR. Elementer er blevet fundet i både...... HPV-16 og p53 3'UTR der eventuelt kan være involveret i post-transkriptionel regulering af messenger RNA (mRNA) kodet af disse gener. Elementerne med konsensus sekvensen UUUUUAU har tidligere været vist at påvirke reguleringen af maternelle mRNA i oogenese og tidlig udvikling. Elementerne kaldes...... cytoplasmatisk polyadenylerings elementer (CPE) da mRNA indeholdende disse bliver deadenyleret efter eksport fra kernen hvorefter de ligger inaktive i cytoplasma, men kan polyadenyleres og blive translationelt aktive. I denne afhandling viser vi at CPE elementer i den tidlige 3'UTR af HPV-16 hæmmer translationen...

  8. A universal strategy for regulating mRNA translation in prokaryotic and eukaryotic cells.

    Science.gov (United States)

    Cao, Jicong; Arha, Manish; Sudrik, Chaitanya; Mukherjee, Abhirup; Wu, Xia; Kane, Ravi S

    2015-04-30

    We describe a simple strategy to control mRNA translation in both prokaryotic and eukaryotic cells which relies on a unique protein-RNA interaction. Specifically, we used the Pumilio/FBF (PUF) protein to repress translation by binding in between the ribosome binding site (RBS) and the start codon (in Escherichia coli), or by binding to the 5' untranslated region of target mRNAs (in mammalian cells). The design principle is straightforward, the extent of translational repression can be tuned and the regulator is genetically encoded, enabling the construction of artificial signal cascades. We demonstrate that this approach can also be used to regulate polycistronic mRNAs; such regulation has rarely been achieved in previous reports. Since the regulator used in this study is a modular RNA-binding protein, which can be engineered to target different 8-nucleotide RNA sequences, our strategy could be used in the future to target endogenous mRNAs for regulating metabolic flows and signaling pathways in both prokaryotic and eukaryotic cells. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. TRPV4 regulates insulin mRNA expression and INS-1E cell death via ERK1/2 and NO-dependent mechanisms.

    Science.gov (United States)

    Billert, M; Skrzypski, M; Sassek, M; Szczepankiewicz, D; Wojciechowicz, T; Mergler, S; Strowski, M Z; Nowak, K W

    2017-07-01

    TRPV4 is a Ca 2+ -permeable, nonselective cation channel. Recently, TRPV4 was implicated in controlling peripheral insulin sensitivity, insulin secretion and apoptosis of pancreatic beta cells. Here, we characterize the role and potential mechanisms of TRPV4 in regulating insulin mRNA expression and cell death in insulin producing INS-1E cells and rat pancreatic islets. TRPV4 protein production was downregulated by siRNA. Intracellular calcium level was measured using Fluo-3 AM. Gene expression was studied by real-time PCR. Phosphorylation of extracellular signal-regulated kinase (ERK1 and ERK2) was detected by Western blot. Nitric oxide (NO) production was assessed by chemiluminescent reaction. Reactive oxygen species (ROS) level was analysed using a fluorogenic dye (DCFDA). Cell death was evaluated by determination of cytoplasmic histone-associated DNA fragments. Downregulation of TRPV4 neither affected insulin mRNA expression nor INS-1E cell growth. By contrast, pharmacological TRPV4 activation by 100nmol/l GSK1016790A increased Ca 2+ levels in INS-1E cells and enhanced insulin mRNA expression after 1 and 3h, whereas a suppression of insulin mRNA expression was detected after 24h incubation. GSK1016790A increased ERK1/2 phosphorylation and NO production but not ROS production. Pharmacological blockade of ERK1/2 attenuated GSK1016790A-induced insulin mRNA expression. Inhibition of NO synthesis by l-NAME failed to affect insulin mRNA expression in GSK1016790A treated INS-1E cells. Furthermore, inhibition of NO production attenuated GSK1016790A-induced INS-1E cell death. In pancreatic islets, 100nmol/l GSK1016790A increased insulin mRNA levels after 3h without inducing cytotoxicity after 24h. In conclusion, TRPV4 differently regulates insulin mRNA expression in INS-1E cells via ERK1/2 and NO-dependent mechanisms. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Light differentially regulates cell division and the mRNA abundance of pea nucleolin during de-etiolation

    Science.gov (United States)

    Reichler, S. A.; Balk, J.; Brown, M. E.; Woodruff, K.; Clark, G. B.; Roux, S. J.

    2001-01-01

    The abundance of plant nucleolin mRNA is regulated during de-etiolation by phytochrome. A close correlation between the mRNA abundance of nucleolin and mitosis has also been previously reported. These results raised the question of whether the effects of light on nucleolin mRNA expression were a consequence of light effects on mitosis. To test this we compared the kinetics of light-mediated increases in cell proliferation with that of light-mediated changes in the abundance of nucleolin mRNA using plumules of dark-grown pea (Pisum sativum) seedlings. These experiments show that S-phase increases 9 h after a red light pulse, followed by M-phase increases in the plumule leaves at 12 h post-irradiation, a time course consistent with separately measured kinetics of red light-induced increases in the expression of cell cycle-regulated genes. These increases in cell cycle-regulated genes are photoreversible, implying that the light-induced increases in cell proliferation are, like nucleolin mRNA expression, regulated via phytochrome. Red light stimulates increases in the mRNA for nucleolin at 6 h post-irradiation, prior to any cell proliferation changes and concurrent with the reported timing of phytochrome-mediated increases of rRNA abundance. After a green light pulse, nucleolin mRNA levels increase without increasing S-phase or M-phase. Studies in animals and yeast indicate that nucleolin plays a significant role in ribosome biosynthesis. Consistent with this function, pea nucleolin can rescue nucleolin deletion mutants of yeast that are defective in rRNA synthesis. Our data show that during de-etiolation, the increased expression of nucleolin mRNA is more directly regulated by light than by mitosis.

  11. Regulation of brain insulin mRNA by glucose and glucagon-like peptide 1.

    Science.gov (United States)

    Madadi, Golnaz; Dalvi, Prasad S; Belsham, Denise D

    2008-11-28

    Whether the brain synthesizes insulin is currently debated. Two clonal, immortalized mouse hypothalamic cell lines from e17, mHypoE-39 and mHypoE-46, express insulin 2 (Ins2), but not Ins1. We analyzed regions necessary for basal gene activity and found that the mouse Ins2 region -110/+183 bp stimulates promoter activity in hypothalamic neurons. The rat Ins2 showed moderate activity, whereas the human promoter construct is repressed below basal levels. In MIN6 pancreatic beta-cells, all of the Ins1 and Ins2 promoter constructs display high levels of transcriptional activity. The cell lines also express components of glucose-sensing machinery and the endogenous glucagon-like peptide 1 receptor (Glp-1R). We observed that 16.7 mM glucose induces Ins2 mRNA, while forskolin and a Glp-1 agonist, exendin-4, induce a biphasic Ins2 mRNA response in mHypoE-39 neurons. The insulin cis-regulatory regions differ between the pancreas and the hypothalamus, and glucose and Glp-1 regulate the expression of hypothalamic insulin.

  12. The pokeweed leaf mRNA transcriptome and its regulation by jasmonic acid.

    Directory of Open Access Journals (Sweden)

    Kira C.M. Neller

    2016-03-01

    Full Text Available The American pokeweed plant, Phytolacca americana, is recognized for synthesizing pokeweed antiviral protein (PAP, a ribosome inactivating protein (RIP that inhibits the replication of several plant and animal viruses. The plant is also a heavy metal accumulator with applications in soil remediation. However, little is known about pokeweed stress responses, as large-scale sequencing projects have not been performed for this species. Here, we sequenced the mRNA transcriptome of pokeweed in the presence and absence of jasmonic acid (JA, a hormone mediating plant defense. Trinity-based de novo assembly of mRNA from leaf tissue and BLASTx homology searches against public sequence databases resulted in the annotation of 59 096 transcripts. Differential expression analysis identified JA-responsive genes that may be involved in defense against pathogen infection and herbivory. We confirmed the existence of several PAP isoforms and cloned a potentially novel isoform of PAP. Expression analysis indicated that PAP isoforms are differentially responsive to JA, perhaps indicating specialized roles within the plant. Finally, we identified 52 305 natural antisense transcript pairs, four of which comprised PAP isoforms, suggesting a novel form of RIP gene regulation. This transcriptome-wide study of a Phytolaccaceae family member provides a source of new genes that may be involved in stress tolerance in this plant. The sequences generated in our study have been deposited in the SRA database under project # SRP069141.

  13. The Pokeweed Leaf mRNA Transcriptome and Its Regulation by Jasmonic Acid.

    Science.gov (United States)

    Neller, Kira C M; Klenov, Alexander; Hudak, Katalin A

    2016-01-01

    The American pokeweed plant, Phytolacca americana, is recognized for synthesizing pokeweed antiviral protein (PAP), a ribosome inactivating protein (RIP) that inhibits the replication of several plant and animal viruses. The plant is also a heavy metal accumulator with applications in soil remediation. However, little is known about pokeweed stress responses, as large-scale sequencing projects have not been performed for this species. Here, we sequenced the mRNA transcriptome of pokeweed in the presence and absence of jasmonic acid (JA), a hormone mediating plant defense. Trinity-based de novo assembly of mRNA from leaf tissue and BLASTx homology searches against public sequence databases resulted in the annotation of 59 096 transcripts. Differential expression analysis identified JA-responsive genes that may be involved in defense against pathogen infection and herbivory. We confirmed the existence of several PAP isoforms and cloned a potentially novel isoform of PAP. Expression analysis indicated that PAP isoforms are differentially responsive to JA, perhaps indicating specialized roles within the plant. Finally, we identified 52 305 natural antisense transcript pairs, four of which comprised PAP isoforms, suggesting a novel form of RIP gene regulation. This transcriptome-wide study of a Phytolaccaceae family member provides a source of new genes that may be involved in stress tolerance in this plant. The sequences generated in our study have been deposited in the SRA database under project # SRP069141.

  14. Transcriptome kinetics is governed by a genome-wide coupling of mRNA production and degradation: a role for RNA Pol II.

    Directory of Open Access Journals (Sweden)

    Ophir Shalem

    2011-09-01

    Full Text Available Transcriptome dynamics is governed by two opposing processes, mRNA production and degradation. Recent studies found that changes in these processes are frequently coordinated and that the relationship between them shapes transcriptome kinetics. Specifically, when transcription changes are counter-acted with changes in mRNA stability, transient fast-relaxing transcriptome kinetics is observed. A possible molecular mechanism underlying such coordinated regulation might lay in two RNA polymerase (Pol II subunits, Rpb4 and Rpb7, which are recruited to mRNAs during transcription and later affect their degradation in the cytoplasm. Here we used a yeast strain carrying a mutant Pol II which poorly recruits these subunits. We show that this mutant strain is impaired in its ability to modulate mRNA stability in response to stress. The normal negative coordinated regulation is lost in the mutant, resulting in abnormal transcriptome profiles both with respect to magnitude and kinetics of responses. These results reveal an important role for Pol II, in regulation of both mRNA synthesis and degradation, and also in coordinating between them. We propose a simple model for production-degradation coupling that accounts for our observations. The model shows how a simple manipulation of the rates of co-transcriptional mRNA imprinting by Pol II may govern genome-wide transcriptome kinetics in response to environmental changes.

  15. Regulation of activity-regulated cytoskeleton protein (Arc) mRNA after acute and chronic electroconvulsive stimulation in the rat

    DEFF Research Database (Denmark)

    Larsen, M H; Olesen, M; Woldbye, D P D

    2005-01-01

    and it would be predicted that gene products involved in antidepressant effects accumulate after repeated ECS. However, repeated ECS reduced Arc gene expression in the CA1 24 h after the last stimulus. These results indicate that Arc is an immediate early gene product regulated by an acute excitatory stimulus...

  16. The p53 target Wig-1 regulates p53 mRNA stability through an AU-rich element

    DEFF Research Database (Denmark)

    Vilborg, Anna; Glahder, Jacob-Andreas Harald; Wilhelm, Margareta T

    2009-01-01

    The p53 target gene Wig-1 encodes a double-stranded-RNA-binding zinc finger protein. We show here that Wig-1 binds to p53 mRNA and stabilizes it through an AU-rich element (ARE) in the 3' UTR of the p53 mRNA. This effect is mirrored by enhanced p53 protein levels in both unstressed cells and cells...... exposed to p53-activating stress agents. Thus, the p53 target Wig-1 is a previously undescribed ARE-regulating protein that acts as a positive feedback regulator of p53, with implications both for the steady-state levels of p53 and for the p53 stress response. Our data reveal a previously undescribed link...... between the tumor suppressor p53 and posttranscriptional gene regulation via AREs in mRNA....

  17. Arsenic Induces Polyadenylation of Canonical Histone mRNA by Down-regulating Stem-Loop-binding Protein Gene Expression*

    Science.gov (United States)

    Brocato, Jason; Fang, Lei; Chervona, Yana; Chen, Danqi; Kiok, Kathrin; Sun, Hong; Tseng, Hsiang-Chi; Xu, Dazhong; Shamy, Magdy; Jin, Chunyuan; Costa, Max

    2014-01-01

    The replication-dependent histone genes are the only metazoan genes whose messenger RNA (mRNA) does not terminate with a poly(A) tail at the 3′-end. Instead, the histone mRNAs display a stem-loop structure at their 3′-end. Stem-loop-binding protein (SLBP) binds the stem-loop and regulates canonical histone mRNA metabolism. Here we report that exposure to arsenic, a carcinogenic metal, decreased cellular levels of SLBP by inducing its proteasomal degradation and inhibiting SLBP transcription via epigenetic mechanisms. Notably, arsenic exposure dramatically increased polyadenylation of canonical histone H3.1 mRNA possibly through down-regulation of SLBP expression. The polyadenylated H3.1 mRNA induced by arsenic was not susceptible to normal degradation that occurs at the end of S phase, resulting in continued presence into mitosis, increased total H3.1 mRNA, and increased H3 protein levels. Excess expression of canonical histones have been shown to increase sensitivity to DNA damage as well as increase the frequency of missing chromosomes and induce genomic instability. Thus, polyadenylation of canonical histone mRNA following arsenic exposure may contribute to arsenic-induced carcinogenesis. PMID:25266719

  18. The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli.

    Science.gov (United States)

    Esquerré, Thomas; Bouvier, Marie; Turlan, Catherine; Carpousis, Agamemnon J; Girbal, Laurence; Cocaign-Bousquet, Muriel

    2016-04-26

    Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation.

  19. Global SUMO proteome responses guide gene regulation, mRNA biogenesis, and plant stress responses

    Directory of Open Access Journals (Sweden)

    Magdalena eMazur

    2012-09-01

    Full Text Available Small-ubiquitin-like MOdifier (SUMO is a key regulator of abiotic stress, disease resistance and development in plants. The identification of >350 plant SUMO targets has revealed many processes modulated by SUMO and potential consequences of SUMO on its targets. Importantly, highly related proteins are SUMO-modified in plants, yeast, and metazoans. Overlapping SUMO targets include heat-shock proteins, transcription regulators, histones, histone-modifying enzymes, proteins involved in DNA damage repair, but also proteins involved in mRNA biogenesis and nucleo-cytoplasmic transport. Proteomics studies indicate key roles for SUMO in gene repression by controlling histone (deacetylation activity at genomic loci. The responsible heavily sumoylated transcriptional repressor complexes are recruited by EAR (Ethylene-responsive element binding factor [ERF]-associated Amphiphilic Repression-motif containing transcription factors in plants. These transcription factors are not necessarily themselves a SUMO target. Conversely, SUMO acetylation prevents binding of downstream partners by preventing binding of SIMs (SUMO-interaction peptide motifs presents in these partners, while SUMO acetylation has emerged as mechanism to recruit specifically bromodomains; bromodomain are generally linked with gene activation. These findings strengthen the idea of a bidirectional sumo-/acetylation switch in gene regulation. Quantitative proteomics has highlighted that global sumoylation provides a dynamic response to protein damage involving SUMO chain-mediated protein degradation, but also SUMO E3 ligase-dependent transcription of HSP (Heat-shock protein genes. With these insights in SUMO function and novel technical advancements, we can now study SUMO dynamics in responses to (abiotic stress in plants.

  20. 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.

  1. A G-rich element forms a G-quadruplex and regulates BACE1 mRNA alternative splicing.

    Science.gov (United States)

    Fisette, Jean-François; Montagna, Daniel R; Mihailescu, Mihaela-Rita; Wolfe, Michael S

    2012-06-01

    β-Site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is the transmembrane aspartyl protease that catalyzes the first cleavage step in the proteolysis of the APP to the amyloid β-protein (Aβ), a process involved in the pathogenesis of Alzheimer disease. BACE1 pre-mRNA undergoes complex alternative splicing, the regulation of which is not well understood. We identified a G-rich sequence within exon 3 of BACE1 involved in controlling splice site selection. Mutation of the G-rich sequence decreased use of the normal 5' splice site of exon 3, which leads to full-length and proteolytically active BACE1, and increased use of an alternative splice site, which leads to a shorter, essentially inactive isoform. Nuclease protection assays, nuclear magnetic resonance, and circular dichroism spectroscopy revealed that this sequence folds into a G-quadruplex structure. Several proteins were identified as capable of binding to the G-rich sequence, and one of these, heterogeneous nuclear ribonucleoprotein H, was found to regulate BACE1 exon 3 alternative splicing and in a manner dependent on the G-rich sequence. Knockdown of heterogeneous nuclear ribonucleoprotein H led to a decrease in the full-length BACE1 mRNA isoform as well as a decrease in Aβ production from APP, suggesting new possibilities for therapeutic approaches to Alzheimer's disease. © 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.

  2. Drosophila Syncrip binds the gurken mRNA localisation signal and regulates localised transcripts during axis specification

    Directory of Open Access Journals (Sweden)

    Suzanne M. McDermott

    2012-04-01

    In the Drosophila oocyte, mRNA transport and localised translation play a fundamental role in axis determination and germline formation of the future embryo. gurken mRNA encodes a secreted TGF-α signal that specifies dorsal structures, and is localised to the dorso-anterior corner of the oocyte via a cis-acting 64 nucleotide gurken localisation signal. Using GRNA chromatography, we characterised the biochemical composition of the ribonucleoprotein complexes that form around the gurken mRNA localisation signal in the oocyte. We identified a number of the factors already known to be involved in gurken localisation and translational regulation, such as Squid and Imp, in addition to a number of factors with known links to mRNA localisation, such as Me31B and Exu. We also identified previously uncharacterised Drosophila proteins, including the fly homologue of mammalian SYNCRIP/hnRNPQ, a component of RNA transport granules in the dendrites of mammalian hippocampal neurons. We show that Drosophila Syncrip binds specifically to gurken and oskar, but not bicoid transcripts. The loss-of-function and overexpression phenotypes of syncrip in Drosophila egg chambers show that the protein is required for correct grk and osk mRNA localisation and translational regulation. We conclude that Drosophila Syncrip is a new factor required for localisation and translational regulation of oskar and gurken mRNA in the oocyte. We propose that Syncrip/SYNCRIP is part of a conserved complex associated with localised transcripts and required for their correct translational regulation in flies and mammals.

  3. Concordant regulation of translation and mRNA abundance for hundreds of targets of a human microRNA.

    Directory of Open Access Journals (Sweden)

    David G Hendrickson

    2009-11-01

    Full Text Available MicroRNAs (miRNAs regulate gene expression posttranscriptionally by interfering with a target mRNA's translation, stability, or both. We sought to dissect the respective contributions of translational inhibition and mRNA decay to microRNA regulation. We identified direct targets of a specific miRNA, miR-124, by virtue of their association with Argonaute proteins, core components of miRNA effector complexes, in response to miR-124 transfection in human tissue culture cells. In parallel, we assessed mRNA levels and obtained translation profiles using a novel global approach to analyze polysomes separated on sucrose gradients. Analysis of translation profiles for approximately 8,000 genes in these proliferative human cells revealed that basic features of translation are similar to those previously observed in rapidly growing Saccharomyces cerevisiae. For approximately 600 mRNAs specifically recruited to Argonaute proteins by miR-124, we found reductions in both the mRNA abundance and inferred translation rate spanning a large dynamic range. The changes in mRNA levels of these miR-124 targets were larger than the changes in translation, with average decreases of 35% and 12%, respectively. Further, there was no identifiable subgroup of mRNA targets for which the translational response was dominant. Both ribosome occupancy (the fraction of a given gene's transcripts associated with ribosomes and ribosome density (the average number of ribosomes bound per unit length of coding sequence were selectively reduced for hundreds of miR-124 targets by the presence of miR-124. Changes in protein abundance inferred from the observed changes in mRNA abundance and translation profiles closely matched changes directly determined by Western analysis for 11 of 12 proteins, suggesting that our assays captured most of miR-124-mediated regulation. These results suggest that miRNAs inhibit translation initiation or stimulate ribosome drop-off preferentially near the

  4. Conserved regions of the DMD 3' UTR regulate translation and mRNA abundance in cultured myotubes.

    Science.gov (United States)

    Larsen, C Aaron; Howard, Michael T

    2014-08-01

    Duchenne muscular dystrophy (DMD), a severe muscle-wasting disease, is caused by mutations in the DMD gene, which encodes for the protein dystrophin. Its regulation is of therapeutic interest as even small changes in expression of functional dystrophin can significantly impact the severity of DMD. While tissue-specific distribution and transcriptional regulation of several DMD mRNA isoforms has been well characterized, the post-transcriptional regulation of dystrophin synthesis is not well understood. Here, we utilize qRTPCR and a quantitative dual-luciferase reporter assay to examine the effects of isoform specific DMD 5' UTRs and the highly conserved DMD 3' UTR on mRNA abundance and translational control of gene expression in C2C12 cells. The 5' UTRs were shown to initiate translation with low efficiency in both myoblasts and myotubes. Whereas, two large highly conserved elements in the 3' UTR, which overlap the previously described Lemaire A and D regions, increase mRNA levels and enhance translation upon differentiation of myoblasts into myotubes. The results presented here implicate an important role for DMD UTRs in dystrophin expression and delineate the cis-acting elements required for the myotube-specific regulation of steady-state mRNA levels and translational enhancer activity found in the DMD 3' UTR. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Conserved regions of the DMD 3’ UTR regulate translation and mRNA abundance in cultured myotubes

    Science.gov (United States)

    Larsen, C. Aaron; Howard, Michael T.

    2014-01-01

    Duchenne muscular dystrophy (DMD), a severe muscle-wasting disease, is caused by mutations in the DMD gene, which encodes for the protein dystrophin. Its regulation is of therapeutic interest as even small changes in expression of functional dystrophin can significantly impact the severity of DMD. While tissue-specific distribution and transcriptional regulation of several DMD mRNA isoforms has been well characterized, the post-transcriptional regulation of dystrophin synthesis is not well understood. Here, we utilize qRTPCR and a quantitative dual-luciferase reporter assay to examine the effects of isoform specific DMD 5’ UTRs and the highly conserved DMD 3’ UTR on mRNA abundance and translational control of gene expression in C2C12 cells. The 5’ UTRs were shown to initiate translation with low efficiency in both myoblasts and myotubes. Whereas, two large highly conserved elements in the 3’ UTR, which overlap the previously described Lemaire A and D regions, increase mRNA levels and enhance translation upon differentiation of myoblasts into myotubes. The results presented here implicate an important role for DMD UTRs in dystrophin expression and delineate the cis-acting elements required for the myotube-specific regulation of steady-state mRNA levels and translational enhancer activity found in the DMD 3’ UTR. PMID:24928536

  6. Bicarbonate-regulated soluble adenylyl cyclase (sAC) mRNA expression and activity in peripheral chemoreceptors.

    Science.gov (United States)

    Nunes, A R; Monteiro, E C; Johnson, S M; Gauda, E B

    2009-01-01

    Peripheral arterial chemoreceptors in the carotid body (CB) are modulated by pH/CO(2). Soluble adenylyl cyclase (sAC) is directly stimulated by bicarbonate ions (HCO(3)). Because CO(2)/HCO(3) mediates depolarization in chemoreceptors, we hypothesized that sAC mRNA would be expressed in the CB, and its expression and function would be regulated by CO(2)/HCO(3).Sprague-Dawley rats at postnatal days 16-17 were used to compare sAC mRNA gene expression between CB and non-chemosensitive tissues: superior cervical (SCG), petrosal (PG) and nodose ganglia (NG) by quantitative real time-PCR. Rat sAC gene expression was standardized to the expression of GAPDH (housekeeping gene) and the data were analyzed with the Pfaffl method. Gene and protein expression, and sAC regulation in the testis was used as a positive control. To determine the regulation of sAC mRNA expression and activity, all tissues were exposed to increasing concentrations of bicarbonate (0, 24, 44 mM, titrated with CO(2) and maintained a constant pH of 7.40). sAC mRNA expression was between 2-11% of CB expression in the SCG, PG and NG. Furthermore, only in the CB did HCO(3) upregulate sAC gene expression and increase cAMP levels. sAC mRNA and protein expression is present in peripheral arterial chemoreceptors and non-chemoreceptors. In the CB, CO(2)/HCO(3) not only activated sAC but also regulated its expression, suggesting that sAC may be involved in the regulation of cAMP levels in response to hyper/hypocapnia.

  7. RNase L controls terminal adipocyte differentiation, lipids storage and insulin sensitivity via CHOP10 mRNA regulation

    DEFF Research Database (Denmark)

    Fabre, Odile Martine Julie; Salehzada, T; Lambert, K

    2012-01-01

    Adipose tissue structure is altered during obesity, leading to deregulation of whole-body metabolism. Its function depends on its structure, in particular adipocytes number and differentiation stage. To better understand the mechanisms regulating adipogenesis, we have investigated the role...... RNase L in RNase L(-/-)-MEFs triggers CHOP10 mRNA instability, allowing increased lipids storage, insulin response and glucose uptake. Similarly, downregulation of CHOP10 mRNA with CHOP10 siRNA in RNase L(-/-)-MEFs improves their differentiation in adipocyte. In vivo, aged RNase L(-)/(-) mice present...

  8. A novel actin mRNA splice variant regulates ACTG1 expression.

    Directory of Open Access Journals (Sweden)

    Meghan C Drummond

    Full Text Available Cytoplasmic actins are abundant, ubiquitous proteins in nucleated cells. However, actin expression is regulated in a tissue- and development-specific manner. We identified a novel cytoplasmic-γ-actin (Actg1 transcript that includes a previously unidentified exon (3a. Inclusion of this exon introduces an in-frame termination codon. We hypothesized this alternatively-spliced transcript down-regulates γ-actin production by targeting these transcripts for nonsense-mediated decay (NMD. To address this, we investigated conservation between mammals, tissue-specificity in mice, and developmental regulation using C2C12 cell culture. Exon 3a is 80% similar among mammals and varies in length from 41 nucleotides in humans to 45 in mice. Though the predicted amino acid sequences are not similar between all species, inclusion of exon 3a consistently results in the in the introduction of a premature termination codon within the alternative Actg1 transcript. Of twelve tissues examined, exon 3a is predominantly expressed in skeletal muscle, cardiac muscle, and diaphragm. Splicing to include exon 3a is concomitant with previously described down-regulation of Actg1 in differentiating C2C12 cells. Treatment of differentiated C2C12 cells with an inhibitor of NMD results in a 7-fold increase in exon 3a-containing transcripts. Therefore, splicing to generate exon 3a-containing transcripts may be one component of Actg1 regulation. We propose that this post-transcriptional regulation occurs via NMD, in a process previously described as "regulated unproductive splicing and translation" (RUST.

  9. G quadruplex RNA structures in PSD-95 mRNA: potential regulators of miR-125a seed binding site accessibility.

    Science.gov (United States)

    Stefanovic, Snezana; Bassell, Gary J; Mihailescu, Mihaela Rita

    2015-01-01

    Fragile X syndrome (FXS) is the most common inherited form of intellectual disability caused by the CGG trinucleotide expansion in the 3'-untranslated region of the FMR1 gene on the X chromosome, that silences the expression of the Fragile X mental retardation protein (FMRP). FMRP has been shown to bind to a G-rich region within the PSD-95 mRNA which encodes for the postsynaptic density protein 95 (PSD-95), and together with the microRNA miR-125a, to play an important role in the reversible inhibition of the PSD-95 mRNA translation in neurons. The loss of FMRP in Fmr1 KO mice disables this translation control in the production of the PSD-95 protein. Interestingly, the miR-125a binding site on PSD-95 mRNA is embedded in the G-rich region bound by FMRP and postulated to adopt one or more G quadruplex structures. In this study, we have used different biophysical techniques to validate and characterize the formation of parallel G quadruplex structures and binding of miR-125a to its complementary sequence located within the 3' UTR of PSD-95 mRNA. Our results indicate that the PSD-95 mRNA G-rich region folds into alternate G quadruplex conformations that coexist in equilibrium. miR-125a forms a stable complex with PSD-95 mRNA, as evident by characteristic Watson-Crick base-pairing that coexists with one of the G quadruplex forms, suggesting a novel mechanism for G quadruplex structures to regulate the access of miR-125a to its binding site. © 2014 Stefanovic et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  10. Effect of insulin on human skeletal muscle mitochondrial ATP production, protein synthesis, and mRNA transcripts

    Science.gov (United States)

    Stump, Craig S.; Short, Kevin R.; Bigelow, Maureen L.; Schimke, Jill M.; Sreekumaran Nair, K.

    2003-06-01

    Mitochondria are the primary site of skeletal muscle fuel metabolism and ATP production. Although insulin is a major regulator of fuel metabolism, its effect on mitochondrial ATP production is not known. Here we report increases in vastus lateralis muscle mitochondrial ATP production capacity (32-42%) in healthy humans (P growth hormone. Increased ATP production occurred in association with increased mRNA levels from both mitochondrial (NADH dehydrogenase subunit IV) and nuclear [cytochrome c oxidase (COX) subunit IV] genes (164-180%) encoding mitochondrial proteins (P muscle mitochondrial protein synthesis, and COX and citrate synthase enzyme activities were increased by insulin (P muscle mitochondrial ATP production for people with type 2 diabetes mellitus, whereas matched nondiabetic controls increased 16-26% (P muscle along with synthesis of gene transcripts and mitochondrial protein in human subjects. Skeletal muscle of type 2 diabetic patients has a reduced capacity to increase ATP production with high insulin levels. cytochrome c oxidase | NADH dehydrogenase subunit IV | amino acids | citrate synthase

  11. BORIS/CTCFL mRNA isoform expression and epigenetic regulation in epithelial ovarian cancer

    Science.gov (United States)

    Link, Petra A.; Zhang, Wa; Odunsi, Kunle; Karpf, Adam R.

    2013-01-01

    Cancer germline (CG) genes are normally expressed in germ cells and aberrantly expressed in a variety of cancers; their immunogenicity has led to the widespread development of cancer vaccines targeting these antigens. BORIS/CTCFL is an autosomal CG antigen and promising cancer vaccine target. BORIS is the only known paralog of CTCF, a gene intimately involved in genomic imprinting, chromatin insulation, and nuclear regulation. We have previously shown that BORIS is expressed in epithelial ovarian cancer (EOC) and that its expression coincides with promoter and global DNA hypomethylation. Recently, 23 different BORIS mRNA variants have been described, and have been functionally grouped into six BORIS isoform families (sf1–sf6). In the present study, we have characterized the expression of BORIS isoform families in normal ovary (NO) and EOC, the latter of which were selected to include two groups with widely varying global DNA methylation status. We find selective expression of BORIS isoform families in NO, which becomes altered in EOC, primarily by the activation of BORIS sf1 in EOC. When comparing EOC samples based on methylation status, we find that BORIS sf1 and sf2 isoform families are selectively activated in globally hypomethylated tumors. In contrast, CTCF is downregulated in EOC, and the ratio of BORIS sf1, sf2, and sf6 isoform families as a function of CTCF is elevated in hypomethylated tumors. Finally, the expression of all BORIS isoform families was induced to varying extents by epigenetic modulatory drugs in EOC cell lines, particularly when DNMT and HDAC inhibitors were used in combination. PMID:23390377

  12. Regulated mRNA Decay in Arabidopsis: A global analysis of differential control by hormones and the circadian clock

    Energy Technology Data Exchange (ETDEWEB)

    Green, Pamela J. [Univ. of Delaware, Newark, DE (United States)

    2010-03-18

    The long-term goal of this research was to better understand the influence of mRNA stability on gene regulation, particularly in response to hormones and the circadian clock. The primary aim of this project was to examine this using DNA microarrays, small RNA analysis and other approaches. We accomplished these objectives, although we were only able to detect small changes in mRNA stability in response to these stimuli. However, the work also contributed to a major breakthrough allowing the identification of small RNAs on a genomic scale in eukaryotes. Moreover, the project prompted us to develop a new way to analyze mRNA decay genome wide. Thus, the research was hugely successful beyond our objectives.

  13. Visfatin mRNA expression in human subcutaneous adipose tissue is regulated by exercise

    DEFF Research Database (Denmark)

    Frydelund-Larsen, Lone; Åkerström, Thorbjörn; Nielsen, Søren

    2006-01-01

    Visfatin [pre-beta-cell colony-enhancing factor (PBEF)] is a novel adipokine that is produced by adipose tissue, skeletal muscle, and liver and has insulin-mimetic actions. Regular exercise enhances insulin sensitivity. In the present study, we therefore examined visfatin mRNA expression...... by elevated levels of plasma visfatin. Recombinant human IL-6 infusion to mimic the exercise-induced IL-6 response (n = 6) had no effect on visfatin mRNA expression in adipose tissue compared with the effect of placebo infusion (n = 6). The finding that exercise enhances subcutaneous adipose tissue visfatin mRNA...... in abdominal subcutaneous adipose tissue and skeletal muscle biopsies obtained from healthy young men at time points 0, 3, 4.5, 6, 9, and 24 h in relation to either 3 h of ergometer cycle exercise at 60% of Vo(2 max) or rest. Adipose tissue visfatin mRNA expression increased threefold at the time points 3, 4...

  14. mRNA expression of genes regulating lipid metabolism in ringed seals (Pusa hispida) from differently polluted areas

    Energy Technology Data Exchange (ETDEWEB)

    Castelli, Martina Galatea [Norwegian Polar Institute, Fram Centre, 9296 Tromsø (Norway); University of Bergen, Department of Biology, 5020 Bergen (Norway); Rusten, Marte; Goksøyr, Anders [University of Bergen, Department of Biology, 5020 Bergen (Norway); Routti, Heli, E-mail: heli.routti@npolar.no [Norwegian Polar Institute, Fram Centre, 9296 Tromsø (Norway)

    2014-01-15

    Highlights: •Genes regulating lipid metabolism were studied in ringed seals. •We compared highly contaminated Baltic seals and less contaminated Svalbard seals. •mRNA expression of hepatic PPARγ was higher in the Baltic seals. •mRNA expression of adipose PPARγ target genes was higher in the Baltic seals. •Contaminant exposure may affect lipid metabolism in the Baltic ringed seals. -- Abstract: There is a growing concern about the ability of persistent organic pollutants (POPs) to influence lipid metabolism. Although POPs are found at high concentrations in some populations of marine mammals, for example in the ringed seal (Pusa hispida) from the Baltic Sea, little is known about the effects of POPs on their lipid metabolism. An optimal regulation of lipid metabolism is crucial for ringed seals during the fasting/molting season. This is a physiologically stressful period, during which they rely on the energy stored in their fat reserves. The mRNA expression levels for seven genes involved in lipid metabolism were analyzed in liver and/or blubber tissue from molting ringed seals from the polluted Baltic Sea and a less polluted reference location, Svalbard (Norway). mRNA expression of genes encoding peroxisome proliferator-activated receptors (PPAR) α and γ and their target genes acyl-coenzyme A oxidase 1 (ACOX1) and cluster of differentiation 36 (CD36) were analyzed in liver. mRNA expression level of genes encoding PPARβ, PPARγ and their target genes encoding fatty acid binding protein 4 (FABP4) and adiponectin (ADIPOQ) were measured in inner and middle blubber layers. In addition, we evaluated the influence of molting status on hepatic mRNA expression of genes encoding PPARs and their target genes in ringed seals from Svalbard. Our results show higher mRNA expression of genes encoding hepatic PPARγ and adipose PPARβ, FABP4, and ADIPOQ in the Baltic seals compared to the Svalbard seals. A positive relationship between mRNA expressions of genes

  15. To be or not to be: the regulation of mRNA fate as a survival strategy during mammalian hibernation.

    Science.gov (United States)

    Tessier, Shannon N; Storey, Kenneth B

    2014-11-01

    Mammalian hibernators undergo profound behavioral, physiological, and biochemical changes in order to cope with hypothermia, ischemia-reperfusion, and finite fuel reserves over days or weeks of continuous torpor. Against a backdrop of global reductions in energy-expensive processes such as transcription and translation, a subset of genes/proteins are strategically upregulated in order to meet challenges associated with hibernation. Consequently, hibernation involves substantial transcriptional and posttranscriptional regulatory mechanisms and provides a phenomenon with which to understand how a set of common genes/proteins can be differentially regulated in order to enhance stress tolerance beyond that which is possible for nonhibernators. The present review focuses on the involvement of messenger RNA (mRNA) interacting factors that play a role in the regulation of gene/protein expression programs that define the hibernating phenotype. These include proteins involved in mRNA processing (i.e., capping, splicing, and polyadenylation) and the possible role of alternative splicing as a means of enhancing protein diversity. Since the total pool of mRNA remains constant throughout torpor, mechanisms which enhance mRNA stability are discussed in the context of RNA binding proteins and mRNA decay pathways. Furthermore, mechanisms which control the global reduction of cap-dependent translation and the involvement of internal ribosome entry sites in mRNAs encoding stress response proteins are also discussed. Finally, the concept of regulating each of these factors in discrete subcellular compartments for enhanced efficiency is addressed. The analysis draws on recent research from several well-studied mammalian hibernators including ground squirrels, bats, and bears.

  16. Triple layered rotavirus VLP production: kinetics of vector replication, mRNA stability and recombinant protein production.

    Science.gov (United States)

    Vieira, Helena L A; Estêvão, Catarina; Roldão, António; Peixoto, Cristina C; Sousa, Marcos F Q; Cruz, Pedro E; Carrondo, Manuel J T; Alves, Paula M

    2005-10-17

    Rotavirus infection causes diarrhoeal disease in infants, killing more than half million children each year. Virus-like particles (VLP) seem to be excellent vaccine candidates, since they are cheaper to produce than attenuated viral vaccines and safer, as they do not contain genetic material. The present work focus on a triple layered particle composed by three rotavirus structural proteins: VP2, VP6 and VP7, produced in an insect cell/baculovirus expressing system. Two strategies were evaluated for 2/6/7 VLP production: co-infection with three monocistronic baculovirus vectors or single-infection with a tricistronic multi-gene baculovirus vector; these strategies were followed at different levels: baculovirus DNA replication kinetics, mRNA stability, protein production and VLP formation. This study highlights some of the reasons why the tricistronic baculovirus strategy is more efficient for production of triple layered rotavirus 2/6/7 VLP than monocistronic co-infection, in particular: (i) the tricistronic vector presents higher DNA replication rates than the monocistronic vectors, (ii) the mRNA stability is invariant for all mRNAs corresponding to VP2, VP6 and VP7 and (iii) the tricistronic baculovirus strategy produces an excess of VP7 over VP6 when compared to the VP7/VP6 stoichiometric ratio in the native rotavirus. Although the co-infection strategy leads to protein production akin to the rotavirus VP7/VP6 stoichiometric ratio, the tricistronic vector strategy yields higher amounts of rotavirus-like particles.

  17. Metabolic regulation of ApoB mRNA editing is associated with phosphorylation of APOBEC-1 complementation factor

    Science.gov (United States)

    Lehmann, David M.; Galloway, Chad A.; Sowden, Mark P.; Smith, Harold C.

    2006-01-01

    Apolipoprotein B (apoB) mRNA editing is a nuclear event that minimally requires the RNA substrate, APOBEC-1 and APOBEC-1 Complementation Factor (ACF). The co-localization of these macro-molecules within the nucleus and the modulation of hepatic apoB mRNA editing activity have been described following a variety of metabolic perturbations, but the mechanism that regulates editosome assembly is unknown. APOBEC-1 was effectively co-immunoprecipitated with ACF from nuclear, but not cytoplasmic extracts. Moreover, alkaline phosphatase treatment of nuclear extracts reduced the amount of APOBEC-1 co-immunoprecipitated with ACF and inhibited in vitro editing activity. Ethanol stimulated apoB mRNA editing was associated with a 2- to 3-fold increase in ACF phosphorylation relative to that in control primary hepatocytes. Significantly, phosphorylated ACF was restricted to nuclear extracts where it co-sedimented with 27S editing competent complexes. Two-dimensional phosphoamino acid analysis of ACF immunopurified from hepatocyte nuclear extracts demonstrated phosphorylation of serine residues that was increased by ethanol treatment. Inhibition of protein phosphatase I, but not PPIIA or IIB, stimulated apoB mRNA editing activity coincident with enhanced ACF phosphorylation in vivo. These data demonstrate that ACF is a metabolically regulated phosphoprotein and suggest that this post-translational modification increases hepatic apoB mRNA editing activity by enhancing ACF nuclear localization/retention, facilitating the interaction of ACF with APOBEC-1 and thereby increasing the probability of editosome assembly and activity. PMID:16820530

  18. G-quadruplexes regulate Epstein-Barr virus-encoded nuclear antigen 1 mRNA translation.

    Science.gov (United States)

    Murat, Pierre; Zhong, Jie; Lekieffre, Lea; Cowieson, Nathan P; Clancy, Jennifer L; Preiss, Thomas; Balasubramanian, Shankar; Khanna, Rajiv; Tellam, Judy

    2014-05-01

    Viruses that establish latent infections have evolved unique mechanisms to avoid host immune recognition. Maintenance proteins of these viruses regulate their synthesis to levels sufficient for maintaining persistent infection but below threshold levels for host immune detection. The mechanisms governing this finely tuned regulation of viral latency are unknown. Here we show that mRNAs encoding gammaherpesviral maintenance proteins contain within their open reading frames clusters of unusual structural elements, G-quadruplexes, which are responsible for the cis-acting regulation of viral mRNA translation. By studying the Epstein-Barr virus-encoded nuclear antigen 1 (EBNA1) mRNA, we demonstrate that destabilization of G-quadruplexes using antisense oligonucleotides increases EBNA1 mRNA translation. In contrast, pretreatment with a G-quadruplex-stabilizing small molecule, pyridostatin, decreases EBNA1 synthesis, highlighting the importance of G-quadruplexes within virally encoded transcripts as unique regulatory signals for translational control and immune evasion. Furthermore, these findings suggest alternative therapeutic strategies focused on targeting RNA structure within viral ORFs.

  19. Changes in mRNA expression of arcuate nucleus appetite-regulating peptides during lactation in rats

    Science.gov (United States)

    Suzuki, Yoshihiro; Nakahara, Keiko; Maruyama, Keisuke; Okame, Rieko; Ensho, Takuya; Inoue, Yoshiyuki; Murakami, Noboru

    2014-01-01

    The contribution of hypothalamic appetite-regulating peptides to further hyperphagia accompanying the course of lactation in rats was investigated by using PCR array and real-time PCR. Furthermore, changes in the mRNA expression for appetite-regulating peptides in the hypothalamic arcuate nucleus (ARC) were analyzed at all stages of pregnancy and lactation, and also after weaning. Food intake was significantly higher during pregnancy, lactation, and after weaning than during non-lactation periods. During lactation, ARC expression of mRNAs for agouti-related protein (AgRP) and peptide YY was increased, whereas that of mRNAs for proopiomelanocortin (POMC) and cholecystokinin (CCK) was decreased, in comparison with non-lactation periods. The increase in AgRP mRNA expression during lactation was especially marked. The plasma level of leptin was significantly decreased during the course of lactation, whereas that of acyl-ghrelin was unchanged. In addition, food intake was negatively correlated with the plasma leptin level during lactation. This study has clarified synchronous changes in the expression of many appetite-regulating peptides in ARC of rats during lactation. Our results suggest that hyperphagia during lactation in rats is caused by decreases in POMC and CCK expression and increases in AgRP expression in ARC, the latter being most notable. Together with the decrease in the blood leptin level, such changes in mRNA expression may explain the further hyperphagia accompanying the course of lactation. PMID:24299740

  20. 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

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

    OpenAIRE

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

    2015-01-01

    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-...

  2. Regulation of adeno-associated virus gene expression in 293 cells: control of mRNA abundance and translation

    Energy Technology Data Exchange (ETDEWEB)

    Trempe, J.P.; Carter, B.J.

    1988-01-01

    The authors studied the effects of the adeno-associated virus (AAV) rep gene on the control of gene expression from the AAV p/sub 40/ promoter in 293 cells in the absence of an adenovirus coinfection. AAV vectors containing the chloramphenicol acetyltransferase (cat) gene were used to measure the levels of cat expression and steady-state mRNA from p/sub 40/. When the rep gene was present in cis or in trans, cat expression from p/sub 40/ was decreased 3- to 10-fold, but there was a 2- to 10-fold increase in the level of p/sub 40/ mRNA. Conversely, cat expression increased and the p/sub 40/ mRNA level decreased in the absence of the rep gene. Both wild-type and carboxyl-terminal truncated Rep proteins were capable of eliciting both effects. These data suggest two roles for the pleiotropic AAV rep gene: as a translational inhibitor and as a positive regulator of p/sub 40/ mRNA levels. They also provide additional evidence for a cis-acting negative regulatory region which decreases RNA from the AAV p/sub 5/ promoter in a fashion independent of rep.

  3. Pumilio binds para mRNA and requires Nanos and Brat to regulate sodium current in Drosophila motoneurons.

    Science.gov (United States)

    Muraro, Nara I; Weston, Andrew J; Gerber, Andre P; Luschnig, Stefan; Moffat, Kevin G; Baines, Richard A

    2008-02-27

    Homeostatic regulation of ionic currents is of paramount importance during periods of synaptic growth or remodeling. Our previous work has identified the translational repressor Pumilio (Pum) as a regulator of sodium current (I(Na)) and excitability in Drosophila motoneurons. In this current study, we show that Pum is able to bind directly the mRNA encoding the Drosophila voltage-gated sodium channel paralytic (para). We identify a putative binding site for Pum in the 3' end of the para open reading frame (ORF). Characterization of the mechanism of action of Pum, using whole-cell patch clamp and real-time reverse transcription-PCR, reveals that the full-length protein is required for translational repression of para mRNA. Additionally, the cofactor Nanos is essential for Pum-dependent para repression, whereas the requirement for Brain Tumor (Brat) is cell type specific. Thus, Pum-dependent regulation of I(Na) in motoneurons requires both Nanos and Brat, whereas regulation in other neuronal types seemingly requires only Nanos but not Brat. We also show that Pum is able to reduce the level of nanos mRNA and as such identify a potential negative-feedback mechanism to protect neurons from overactivity of Pum. Finally, we show coupling between I(Na) (para) and I(K) (Shal) such that Pum-mediated change in para results in a compensatory change in Shal. The identification of para as a direct target of Pum represents the first ion channel to be translationally regulated by this repressor and the location of the binding motif is the first example in an ORF rather than in the canonical 3'-untranslated region of target transcripts.

  4. PPARg mRNA in the adult mouse hypothalamus: distribution and regulation in response to dietary challenges

    Directory of Open Access Journals (Sweden)

    Yang eLiu

    2015-09-01

    Full Text Available Peroxisome proliferator-activated receptor gamma (PPARg is a ligand-activated transcription factor that was originally identified as a regulator of peroxisome proliferation and adipocyte differentiation. Emerging evidence suggests that functional PPARg signaling also occurs within the hypothalamus. However, the exact distribution and identities of PPARg-expressing hypothalamic cells remains under debate. The present study systematically mapped PPARg mRNA expression in the adult mouse brain using in situ hybridization histochemistry. PPARg mRNA was found to be expressed at high levels outside the hypothalamus including the neocortex, the olfactory bulb, the organ of the vasculosum of the lamina terminalis, and the subfornical organ. Within the hypothalamus, PPARg was present at moderate levels in the suprachiasmatic nucleus and the ependymal of the 3rd ventricle. In all examined feeding-related hypothalamic nuclei, PPARg was expressed at very low levels that were close to the limit of detection. Using qPCR techniques, we demonstrated that PPARg mRNA expression was upregulated in the suprachiasmatic nucleus in response to fasting. Double in situ hybridization further demonstrated that PPARg was primarily expressed in neurons. Collectively, our observations provide a comprehensive map of PPARg distribution and regulation in the intact adult mouse hypothalamus.

  5. Do pyrimidine nucleotides regulate translatability of globin mRNA as purine nucleotides do?

    Science.gov (United States)

    Rubin, H N; Almendarez, E; Halim, M N

    1988-01-01

    1. When rabbit globin mRNA was incubated with rabbit reticulocyte lysate in the presence of various concentrations of nucleotides, globin synthesis was inhibited or stimulated dependent on dose. 2. Pyrimidine nucleotides inhibited protein synthesis at 0.3 mM, whereas 2 mM of purine nucleotides were required to cause similar inhibition. 3. Adenosine mono- and diphosphate inhibited globin synthesis at a concentration of only 1 mM; however, the sequence is AMP greater than ADP greater than ATP. 4. Translation arrest by these nucleotides was instantaneous. 5. These results suggest that these nucleotides may provide a structural component for maintaining the integrity, the conformation of mRNA or of the messenger ribonucleoprotein (mRNP).

  6. 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

    of protein synthesis represents one mechanism used to control the different requirements for myelin sheath at each axo–glia interaction. Prior work has established that β1-integrins are involved in the axoglial interactions that initiate myelination. Here, we show that integrin activation regulates...... 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...... sheath. Furthermore, knockdown of hnRNP-K inhibits MBP protein synthesis during myelination. Together, these results identify a novel pathway by which axoglial adhesion molecules coordinate MBP synthesis with myelin sheath formation...

  7. Ferroportin mRNA is down-regulated in granulosa and cervical cells from infertile women.

    Science.gov (United States)

    Moreno-Navarrete, José Maria; López-Navarro, Eva; Candenas, Luz; Pinto, Francisco; Ortega, Francisco J; Sabater-Masdeu, Mònica; Fernández-Sánchez, Manuel; Blasco, Victor; Romero-Ruiz, Antonio; Fontán, Marina; Ricart, Wifredo; Tena-Sempere, Manuel; Fernández-Real, José M

    2017-01-01

    To explore the relationship between iron and infertility by investigating iron-related gene expression in granulosa and uterine cervical cells. Case-control study. Two tertiary hospitals. Two independent cohorts of fertile (n = 18 and n = 17) and infertile (n = 31 and n = 35) women. In vitro fertilization. Gene expression levels of ferritin light chain (FTL), ferritin heavy chain (FTH), transferrin receptor (TFRC), and ferroportin (SLC40A1) mRNA were analyzed in granulosa and cervical cells. In the first cohort, fertile and infertile women were similar in body mass index. Ferroportin mRNA levels were decreased in granulosa cells from infertile women in parallel with increased serum hepcidin levels. A positive association between ferroportin and TFRC mRNA, a gene associated with intracellular iron deficiency, was observed only in granulosa cells from fertile women. The major findings were replicated in a second independent cohort. Ferroportin mRNAs and circulating hepcidin identify a subset of infertile women and may constitute a target for therapy. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  8. Targeted mRNA oxidation regulates sunflower seed dormancy alleviation during dry after-ripening.

    Science.gov (United States)

    Bazin, Jérémie; Langlade, Nicolas; Vincourt, Patrick; Arribat, Sandrine; Balzergue, Sandrine; El-Maarouf-Bouteau, Hayat; Bailly, Christophe

    2011-06-01

    After-ripening is the mechanism by which dormant seeds become nondormant during their dry storage after harvest. The absence of free water in mature seeds does not allow detectable metabolism; thus, the processes associated with dormancy release under these conditions are largely unknown. We show here that sunflower (Helianthus annuus) seed alleviation of dormancy during after-ripening is associated with mRNA oxidation and that this oxidation is prevented when seeds are maintained dormant. In vitro approaches demonstrate that mRNA oxidation results in artifacts in cDNA-amplified fragment length polymorphim analysis and alters protein translation. The oxidation of transcripts is not random but selective, and, using microarrays, we identified 24 stored mRNAs that became highly oxidized during after-ripening. Oxidized transcripts mainly correspond to genes involved in responses to stress and in cell signaling. Among them, protein phosphatase 2C PPH1, mitogen-activated protein kinase phosphatase 1, and phenyl ammonia lyase 1 were identified. We propose that targeted mRNA oxidation during dry after-ripening of dormant seeds could be a process that governs cell signaling toward germination in the early steps of seed imbibition.

  9. A hairpin within YAP mRNA 3′UTR functions in regulation at post-transcription level

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Yuen; Wang, Yuan; Feng, Jinyan; Feng, Guoxing; Zheng, Minying; Yang, Zhe; Xiao, Zelin; Lu, Zhanping [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China); Ye, Lihong [State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China); Zhang, Xiaodong, E-mail: zhangxd@nankai.edu.cn [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China)

    2015-04-03

    The central dogma of gene expression is that DNA is transcribed into messenger RNAs, which in turn serve as the template for protein synthesis. Recently, it has been reported that mRNAs display regulatory roles that rely on their ability to compete for microRNA binding, independent of their protein-coding function. However, the regulatory mechanism of mRNAs remains poorly understood. Here, we report that a hairpin within YAP mRNA 3′untranslated region (3′UTR) functions in regulation at post-transcription level through generating endogenous siRNAs (esiRNAs). Bioinformatics analysis for secondary structure showed that YAP mRNA displayed a hairpin structure (termed standard hairpin, S-hairpin) within its 3′UTR. Surprisingly, we observed that the overexpression of S-hairpin derived from YAP 3′UTR (YAP-sh) increased the luciferase reporter activities of transcriptional factor NF-κB and AP-1 in 293T cells. Moreover, we identified that a fragment from YAP-sh, an esiRNA, was able to target mRNA 3′UTR of NF2 (a member of Hippo-signaling pathway) and YAP mRNA 3′UTR itself in hepatoma cells. Thus, we conclude that the YAP-sh within YAP mRNA 3′UTR may serve as a novel regulatory element, which functions in regulation at post-transcription level. Our finding provides new insights into the mechanism of mRNAs in regulatory function. - Highlights: • An S-hairpin within YAP mRNA 3′UTR possesses regulatory function. • YAP-sh acts as a regulatory element for YAP at post-transcription level. • YAP-sh-3p20, an esiRNA derived from YAP-sh, targets mRNAs of YAP and NF2. • YAP-sh-3p20 depresses the proliferation of HepG2 cells in vitro.

  10. Fasting and exercise differentially regulate BDNF mRNA expression in human skeletal muscle.

    Science.gov (United States)

    Walsh, Jeremy J; Edgett, Brittany A; Tschakovsky, Michael E; Gurd, Brendon J

    2015-01-01

    Brain-derived neurotrophic factor (BDNF) gene expression was measured in human skeletal muscle following 3 intensities of exercise and a 48-h fast. No change in BDNF mRNA was observed following exercise, while fasting upregulated BDNF by ∼ 3.5-fold. These changes were dissociated from changes in peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) following exercise (+2- to 15-fold) and fasting (∼-25%). These results challenge our understanding of the response of BDNF to energetic stress and highlight the importance of future work in this area.

  11. Regulation of c-Myc mRNA by L11 in Response to UV and Gamma Irradiation

    Science.gov (United States)

    2014-12-01

    L11 mutations are associated with cleft palate and abnormal thumbs in Diamond-Blackfan anemia patients. Am. J. Hum. Genet. 83:769–780. 23. Gomez-Roman...24 (Figure 1). We also tested an array of miRNAs possessing tumor suppressor functions for L11 binding miR-16 miR-1248 miR-3944 (-) miR-191 miR...32), and tristetraprolin (TTP) (39), have been found to bind to c-myc AREs and regulate c-myc mRNA sta- bility. To test whether L11 binds to c-myc

  12. Cellular retinoic-acid-binding-protein and retinol-binding-protein mRNA expression in the cells of the rat seminiferous tubules and their regulation by retinoids.

    Science.gov (United States)

    Faraonio, R; Galdieri, M; Colantuoni, V

    1993-02-01

    The levels of the mRNA corresponding to the intracellular binding proteins for retinoic acid and retinol (CRABP1 and CRBP1, respectively) were studied in primary cultures of somatic and germ cells of the rat seminiferous tubules. We show that the CRABP1 mRNA is expressed in Sertoli and germ cells and a single molecular species of mRNA is detected. CRBP1 mRNA is detected in Sertoli and peritubular cells. The regulation of the expression of both genes by retinoids was studied in Sertoli cells. CRABP1 mRNA levels are not affected by either retinoic acid or retinol, whereas both compounds positively regulate CRBP1 mRNA synthesis in a dose-dependent manner. A fivefold increase in CRBP1 mRNA levels was observed 32-48 h after addition of either agent. These results demonstrate that in Sertoli cells the expression of CRABP1 is not affected by retinoids, similar to the situation observed in vivo and in other in-vitro cultures. CRBP1-gene expression is, instead, induced and the variations in CRBP1-mRNA levels may regulate the intracellular concentrations of retinoids, as a response to changes in the vitamin-A nutritional status.

  13. REDUCED NITRIC OXIDE PRODUCTION AND INOS MRNA EXPRESSION IN IFN-G STIMULATED CHICKEN MACROPHAGES TRANSFECTED WITH INOS SIRNAS

    Science.gov (United States)

    Utilizing RNA interference technology with siRNA in the HD-11 macrophage cell line, we determined how the inhibition or knock-down of the iNOS (inducible nitric oxide synthase) gene affected IFN-y' induced macrophage production of nitric oxide (NO) and mRNA expression of genes involved in this biolo...

  14. Global mRNA sequencing of human skeletal muscle: Search for novel exercise-regulated myokines

    Directory of Open Access Journals (Sweden)

    S. Pourteymour

    2017-04-01

    Conclusion: We identified 17 new, exercise-responsive transcripts encoding secretory proteins. We further identified CSF1 as a novel myokine, which is secreted from cultured muscle cells and up-regulated in muscle and plasma after acute exercise.

  15. Dietary glycerol for quail: association between productive performance and COX III mRNA expression.

    Science.gov (United States)

    Silva, S C C; Gasparino, E; Batista, E; Tanamati, F; Vesco, A P D; Lala, B; de Oliveira, D P

    2016-05-25

    This study was carry out to evaluate mRNA expression of mitochondrial cytochrome c oxidase III in the Pectoralis superficialis muscle of 28-day-old quails fed diets containing 0, 8, and 12% glycerol. Total RNA was extracted (N = 10) and cDNA was amplified using specifics primers for qRT-PCR. Feed efficiency and feed intake were evaluated. COX III mRNA expression in breast muscle was higher in the group fed with 12% glycerol (0.863 AU); no differences were observed in the expression of this gene between the muscle of animals fed diets without glycerol (0.357 AU) and 8% glycerol (0.415 AU). Quails that showed greater COX III mRNA expression also showed the lowest feed efficiency. These results show that there is a difference in COX III mRNA expression in breast muscle of 28-day-old quail fed diets different concentrations of glycerol.

  16. Regulation of Egr-1, VIP, and Shh mRNA and Egr-1 protein in the mouse retina by light and image quality.

    Science.gov (United States)

    Brand, Christine; Burkhardt, Eva; Schaeffel, Frank; Choi, Jeong Won; Feldkaemper, Marita Pauline

    2005-04-28

    To analyze mRNA expression changes of Egr-1, VIP, and Shh under different light and treatment conditions in mice. The mRNA expression levels of the three genes and additionally the Egr-1 protein expression were compared in form deprived eyes and eyes with normal vision. Moreover, the influence of dark to light and light to dark transitions and of changes in retinal illumination on mRNA levels was investigated. Form deprivation of mice was induced by fitting frosted diffusers over one eye and an attentuation matched neutral density (ND) filter over the other eye. To measure the effects of retinal illumination changes on mRNA expression, animals were bilaterally fitted with different ND filters. Semiquantitative real-time RT-PCR was used to measure the mRNA levels and immunohistochemistry was applied to localize and detect Egr-1 protein. The expression levels of both Egr-1 mRNA and protein were reduced in form deprived eyes compared to their fellow eyes after 30 min and 1 h, respectively. Egr-1 mRNA was strikingly upregulated both after dark to light and light to dark transitions, whereas minor changes in retinal illumination by covering the eyes with neutral density filters did not alter Egr-1 mRNA expression. In mice, the mRNA levels of VIP and Shh were not affected by form deprivation, but they were found to be regulated depending on the time of day. Both Egr-1 mRNA and protein expression levels were strongly regulated by light, especially by transitions between light and darkness. Image contrast may exert an additional influence on mRNA and protein expression of Egr-1, particularly in the cells in the ganglion cell layer and in bipolar cells.

  17. Regulation of mRNA decay in plant responses to salt and osmotic stress

    NARCIS (Netherlands)

    Kawa, D.; Testerink, C.

    Plant acclimation to environmental stresses requires fast signaling to initiate changes in developmental and metabolic responses. Regulation of gene expression by transcription factors and protein kinases acting upstream are important elements of responses to salt and drought. Gene expression can be

  18. Regulation of mRNA Levels by Decay-Promoting Introns that Recruit the Exosome Specificity Factor Mmi1

    Directory of Open Access Journals (Sweden)

    Cornelia Kilchert

    2015-12-01

    Full Text Available In eukaryotic cells, inefficient splicing is surprisingly common and leads to the degradation of transcripts with retained introns. How pre-mRNAs are committed to nuclear decay is unknown. Here, we uncover a mechanism by which specific intron-containing transcripts are targeted for nuclear degradation in fission yeast. Sequence elements within these “decay-promoting” introns co-transcriptionally recruit the exosome specificity factor Mmi1, which induces degradation of the unspliced precursor and leads to a reduction in the levels of the spliced mRNA. This mechanism negatively regulates levels of the RNA helicase DDX5/Dbp2 to promote cell survival in response to stress. In contrast, fast removal of decay-promoting introns by co-transcriptional splicing precludes Mmi1 recruitment and relieves negative expression regulation. We propose that decay-promoting introns facilitate the regulation of gene expression. Based on the identification of multiple additional Mmi1 targets, including mRNAs, long non-coding RNAs, and sn/snoRNAs, we suggest a general role in RNA regulation for Mmi1 through transcript degradation.

  19. Testosterone Regulates NUCB2 mRNA Expression in Male Mouse Hypothalamus and Pituitary Gland

    OpenAIRE

    Seon, Sojeong; Jeon, Daun; Kim, Heejeong; Chung, Yiwa; Choi, Narae; Yang, Hyunwon

    2017-01-01

    ABSTRACT Nesfatin-1/NUCB2 is known to take part in the control of the appetite and energy metabolism. Recently, many reports have shown nesfatin-1/NUCB2 expression and function in various organs. We previously demonstrated that nesfatin-1/NUCB2 expression level is higher in the pituitary gland compared to other organs and its expression is regulated by 17?-estradiol and progesterone secreted from the ovary. However, currently no data exist on the expression of nesfatin-1/NUCB2 and its regulat...

  20. Hypoxia regulates Notch-3 mRNA and receptor activation in prostate cancer cells

    Directory of Open Access Journals (Sweden)

    Armelle Meunier

    2016-05-01

    Full Text Available The Notch-3 receptor is a recognized key regulator of vascular responses and is increasingly associated with tumorigenesis. Hypoxia-inducible factors activate specific signaling pathways such as Notch in a number of cellular models. This study aimed to evaluate the regulation of Notch-3 by hypoxia in prostate cancer cells. Notch-3 gene and protein expression was established in a panel of aerobic and hypoxic prostate cell lines in vitro, the CWR22 xenograft model and RNA extracted from low grade (Gleason score = 7; non-hypoxic (low HIF, low VEGF; hypoxic (high HIF, high VEGF patient FFPE specimens. NOTCH-3 was upregulated in PC3 (3-fold, 22Rv1 (4.1-fold and DU145 (3.8-fold but downregulated in LnCaP (12-fold compared to the normal cell lines. NOTCH-3 expression was modified following hypoxic exposure in these cells. NOTCH-3 was upregulated (2.2-fold in higher grade and hypoxic tumors, when compared to benign and aerobic pools. In the CWR22 xenograft model, Notch-3 expression was restored in castrate resistant tumors. Nuclear translocation of the Notch-3 intracellular domain was no longer detected following exposure of cells to hypoxia but not associated with a change in expression of HES-1. Our data further identifies Notch-3 as a potentially key hypoxic-responsive member of the Notch pathway in prostate tumorigenesis.

  1. Autogenous translational regulation of the Borna disease virus negative control factor X from polycistronic mRNA using host RNA helicases.

    Directory of Open Access Journals (Sweden)

    Yohei Watanabe

    2009-11-01

    Full Text Available Borna disease virus (BDV is a nonsegmented, negative-strand RNA virus that employs several unique strategies for gene expression. The shortest transcript of BDV, X/P mRNA, encodes at least three open reading frames (ORFs: upstream ORF (uORF, X, and P in the 5' to 3' direction. The X is a negative regulator of viral polymerase activity, while the P phosphoprotein is a necessary cofactor of the polymerase complex, suggesting that the translation of X is controlled rigorously, depending on viral replication. However, the translation mechanism used by the X/P polycistronic mRNA has not been determined in detail. Here we demonstrate that the X/P mRNA autogenously regulates the translation of X via interaction with host factors. Transient transfection of cDNA clones corresponding to the X/P mRNA revealed that the X ORF is translated predominantly by uORF-termination-coupled reinitiation, the efficiency of which is upregulated by expression of P. We found that P may enhance ribosomal reinitiation at the X ORF by inhibition of the interaction of the DEAD-box RNA helicase DDX21 with the 5' untranslated region of X/P mRNA, via interference with its phosphorylation. Our results not only demonstrate a unique translational control of viral regulatory protein, but also elucidate a previously unknown mechanism of regulation of polycistronic mRNA translation using RNA helicases.

  2. Rbfox proteins regulate alternative mRNA splicing through evolutionarily conserved RNA bridges.

    Science.gov (United States)

    Lovci, Michael T; Ghanem, Dana; Marr, Henry; Arnold, Justin; Gee, Sherry; Parra, Marilyn; Liang, Tiffany Y; Stark, Thomas J; Gehman, Lauren T; Hoon, Shawn; Massirer, Katlin B; Pratt, Gabriel A; Black, Douglas L; Gray, Joe W; Conboy, John G; Yeo, Gene W

    2013-12-01

    Alternative splicing (AS) enables programmed diversity of gene expression across tissues and development. We show here that binding in distal intronic regions (>500 nucleotides (nt) from any exon) by Rbfox splicing factors important in development is extensive and is an active mode of splicing regulation. Similarly to exon-proximal sites, distal sites contain evolutionarily conserved GCATG sequences and are associated with AS activation and repression upon modulation of Rbfox abundance in human and mouse experimental systems. As a proof of principle, we validated the activity of two specific Rbfox enhancers in KIF21A and ENAH distal introns and showed that a conserved long-range RNA-RNA base-pairing interaction (an RNA bridge) is necessary for Rbfox-mediated exon inclusion in the ENAH gene. Thus we demonstrate a previously unknown RNA-mediated mechanism for AS control by distally bound RNA-binding proteins.

  3. Regulation of Trypanosoma brucei Total and Polysomal mRNA during Development within Its Mammalian Host.

    Directory of Open Access Journals (Sweden)

    Paul Capewell

    Full Text Available The gene expression of Trypanosoma brucei has been examined extensively in the blood of mammalian hosts and in forms found in the midgut of its arthropod vector, the tsetse fly. However, trypanosomes also undergo development within the mammalian bloodstream as they progress from morphologically 'slender forms' to transmissible 'stumpy forms' through morphological intermediates. This transition is temporally progressive within the first wave of parasitaemia such that gene expression can be monitored in relatively pure slender and stumpy populations as well as during the progression between these extremes. The development also represents the progression of cells from translationally active forms adapted for proliferation in the host to translationally quiescent forms, adapted for transmission. We have used metabolic labelling to quantitate translational activity in slender forms, stumpy forms and in forms undergoing early differentiation to procyclic forms in vitro. Thereafter we have examined the cohort of total mRNAs that are enriched throughout development in the mammalian bloodstream (slender, intermediate and stumpy forms, irrespective of strain, revealing those that exhibit consistent developmental regulation rather than sample specific changes. Transcripts that cosediment with polysomes in stumpy forms and slender forms have also been enriched to identify transcripts that escape translational repression prior to transmission. Combined, the expression and polysomal association of transcripts as trypanosomes undergo development in the mammalian bloodstream have been defined, providing a resource for trypanosome researchers. This facilitates the identification of those that undergo developmental regulation in the bloodstream and therefore those likely to have a role in the survival and capacity for transmission of stumpy forms.

  4. Sam68 regulates EMT through alternative splicing–activated nonsense-mediated mRNA decay of the SF2/ASF proto-oncogene

    Science.gov (United States)

    Valacca, Cristina; Bonomi, Serena; Buratti, Emanuele; Pedrotti, Simona; Baralle, Francisco Ernesto; Sette, Claudio

    2010-01-01

    Epithelial-to-mesenchymal transition (EMT) and its reversal (MET) are crucial cell plasticity programs that act during development and tumor metastasis. We have previously shown that the splicing factor and proto-oncogene SF2/ASF impacts EMT/MET through production of a constitutively active splice variant of the Ron proto-oncogene. Using an in vitro model, we now show that SF2/ASF is also regulated during EMT/MET by alternative splicing associated with the nonsense-mediated mRNA decay pathway (AS-NMD). Overexpression and small interfering RNA experiments implicate the splicing regulator Sam68 in AS-NMD of SF2/ASF transcripts and in the choice between EMT/MET programs. Moreover, Sam68 modulation of SF2/ASF splicing appears to be controlled by epithelial cell–derived soluble factors that act through the ERK1/2 signaling pathway to regulate Sam68 phosphorylation. Collectively, our results reveal a hierarchy of splicing factors that integrate splicing decisions into EMT/MET programs in response to extracellular stimuli. PMID:20876280

  5. Sam68 regulates EMT through alternative splicing-activated nonsense-mediated mRNA decay of the SF2/ASF proto-oncogene.

    Science.gov (United States)

    Valacca, Cristina; Bonomi, Serena; Buratti, Emanuele; Pedrotti, Simona; Baralle, Francisco Ernesto; Sette, Claudio; Ghigna, Claudia; Biamonti, Giuseppe

    2010-10-04

    Epithelial-to-mesenchymal transition (EMT) and its reversal (MET) are crucial cell plasticity programs that act during development and tumor metastasis. We have previously shown that the splicing factor and proto-oncogene SF2/ASF impacts EMT/MET through production of a constitutively active splice variant of the Ron proto-oncogene. Using an in vitro model, we now show that SF2/ASF is also regulated during EMT/MET by alternative splicing associated with the nonsense-mediated mRNA decay pathway (AS-NMD). Overexpression and small interfering RNA experiments implicate the splicing regulator Sam68 in AS-NMD of SF2/ASF transcripts and in the choice between EMT/MET programs. Moreover, Sam68 modulation of SF2/ASF splicing appears to be controlled by epithelial cell-derived soluble factors that act through the ERK1/2 signaling pathway to regulate Sam68 phosphorylation. Collectively, our results reveal a hierarchy of splicing factors that integrate splicing decisions into EMT/MET programs in response to extracellular stimuli.

  6. The Organization of Regulated Production

    DEFF Research Database (Denmark)

    Jansen, Jos; Jeon, Doh-Shin; Menicucci, Domenico

    2008-01-01

    and weak positive (respectively, strong positive) correlation. Second, if the firms can collude under VS and know all costs, then VS is equivalent to VI. However, if firms collude under asymmetric information, then collusion does not affect the choice between VS and VI, since the regulator takes advantage...... of the transaction costs created by asymmetric information....

  7. Regulation of the glucocorticoid receptor mRNA levels in the gills of Atlantic salmon (Salmo salar during smoltification

    Directory of Open Access Journals (Sweden)

    MAZURAIS D.

    1998-07-01

    Full Text Available The regulation of the Glucocorticoid Receptor (GR transcript was investigated in the gills of Atlantic salmon (Salmo salar during the parr-smolt transformation. Sampling of parr and smolt fish was performed between December and July and in particular during the smoltification period occurring in spring. Quantification of GR transcripts revealed differences between the two groups in March and at the beginning of April. During these dates, the amounts of GR mRNA in parr gills were respectively three and two fold lower than those measured in smolts. In order to determine which factors are responsible for these differences, we studied the long-term effects of prolactin and Cortisol treatments on GR transcript in the gills of presmolt fish. The plasma levels of these two hormones respectively drop and rise during smoltification. Contrary to Cortisol long-term treatment which did not modify the amount of gill GR transcript, short-term treatment induced a significant decrease within 12 hours. Prolactin long-term treatment caused a significant increase of GR transcript abundance after 13 days of implant treatment. This result is unexpected with regard to those obtained in the smoltification analysis but is in agreement with previous studies performed in mammary gland revealing a positive control of PRL on GR in epithelial cells. Our data suggest that the regulation of the GR transcript during the parr-smolt transformation probably involves several hormonal factors.

  8. [Two Elovl5-like elongase genes in Cyprinus carpio var. Jian: Gene characterization, mRNA expression, and nutritional regulation].

    Science.gov (United States)

    Ren, H-T; Huang, Y; Tang, Y-K; Yu, J-H; Xu, P

    2015-01-01

    Elovl5 elongase is a critical enzyme involved in the highly unsaturated fatty acid (HUFA) biosynthesis. There is very little information on the evolution and functional characterization of Elovl5-a and Elovl5-b genes in common carp (Cyprinus carpio var. Jian). In the present study, the genomic sequences and structures of two putative Elovl5-like elongase genes in the common carp genome were obtained. The mRNA expression patterns of Elovl5-a and Elovl5-b in tissues, hatching carp embryos, and juveniles under nutritional regulation were investigated. The results show that the two Elovl5 elongase genes have similar organization, coding 8 exons of high identity and introns of distinct size and sequence composition. They are not allelic variants of a single gene. Both Elovl5 elongase genes are highly expressed in liver, intestine (pyloric caeca) and brain. Elovl5-a and Elovl5-b mRNAs showed increased expression from newly hatched to 20 days after hatching. The regulation of Elovl5-a and Elovl5-b in response to dietary fatty acid composition was determined in liver, brain and intestine (pyloric caeca) of common carp fed with diets: (i) fish oil (FO) rich in n-3 HUFA, (ii) corn oil (CO, 18:2n-6) or (iii) linseed oil (LO, 18:3n-3). Also the differential expression of Elovl5-a and Elovl5-b genes in liver, brain and intestine in common carps fed with different oil sources was studied. Further work aimed at the determination of the mechanisms of differential expression of the Elovl5-a and Elovl5-b in different tissues and the roles of transcription factors in regulating HUFA synthesis is in progress.

  9. Effects of antihistamine on up-regulation of histamine H1 receptor mRNA in the nasal mucosa of patients with pollinosis induced by controlled cedar pollen challenge in an environmental exposure unit.

    Science.gov (United States)

    Kitamura, Yoshiaki; Nakagawa, Hideyuki; Fujii, Tatsuya; Sakoda, Takema; Enomoto, Tadao; Mizuguchi, Hiroyuki; Fukui, Hiroyuki; Takeda, Noriaki

    2015-11-01

    In the present study, we examined the effects of antihistamine on the up-regulation of H1R mRNA in the nasal mucosa of patients with pollinosis induced by controlled exposure to pollen using an environmental exposure unit. Out of 20 patients, we designated 14 responders, whose levels of H1R mRNA in the nasal mucosa were increased after the first pollen exposure and excluded 6 non-responders. Accordingly, the first exposure to pollen without treatment significantly induced both nasal symptoms and the up-regulation of H1R mRNA in the nasal mucosa of the responders. Subsequently, prophylactic administration of antihistamine prior to the second pollen exposure significantly inhibited both of the above effects in the responders. Moreover, the nasal expression of H1R mRNA before the second pollen exposure in the responders pretreated with antihistamine was significantly decreased, as compared with that before the first pollen exposure without treatment. These findings suggest that antihistamines suppressed histamine-induced transcriptional activation of H1R gene in the nasal mucosa, in addition to their blocking effect against histamine on H1R, resulting in a decrease of nasal symptoms. These findings further suggest that by their inverse agonistic activity, antihistamines suppress the basal transcription of nasal H1R in the absence of histamine in responders. Copyright © 2015 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.

  10. Nickel ions selectively inhibit lipopolysaccharide-induced interleukin-6 production by decreasing its mRNA stability.

    Directory of Open Access Journals (Sweden)

    Sanki Asakawa

    Full Text Available Nickel (Ni ions easily elute from many alloys and elicit inflammation and allergies. Previous studies have shown that infections due to the implantation of medical devices cause inflammation and enhance the elution of Ni ions (Ni²⁺. However, cross-talk between infection- and Ni²⁺-induced signaling pathways has not yet been elucidated in detail. In the present study, we investigated the effects of Ni2+ on the lipopolysaccharide (LPS-induced production of cytokines in a LPS-induced air pouch-type inflammation model in BALB/c mice and the murine macrophage cell line RAW264. We demonstrated that Ni²⁺ inhibited the LPS-induced production of interleukin (IL-6, but not that of tumor necrosis factor (TNF-α both in vivo and in vitro. This inhibitory effect was also observed with cobalt ion (Co²⁺, but not with chloride ion (Cl⁻, zinc ion (Zn²⁺, or palladium ion (Pd²⁺, and was highly selective to the production of IL-6. Ni²⁺ did not inhibit the activation of ERK1/2, p38 MAPK, or JNK. Although Ni²⁺ decreased IL-6 mRNA levels, it failed to inhibit the LPS-induced activation of the IL-6 promoter. An experiment using actinomycin D, a transcription inhibitor, revealed that Ni²⁺ decreased the stability of IL-6 mRNA. Moreover, Ni²⁺ inhibited the LPS-induced expression of Arid5a, but not regnase-1. These results demonstrated that Ni²⁺ may have selectively inhibited the LPS-induced production of IL-6 by decreasing the Arid5a-dependent stabilization of IL-6 mRNA.

  11. Sub-cellular mRNA localization modulates the regulation of gene expression by small RNAs in bacteria

    Science.gov (United States)

    Teimouri, Hamid; Korkmazhan, Elgin; Stavans, Joel; Levine, Erel

    2017-10-01

    Small non-coding RNAs can exert significant regulatory activity on gene expression in bacteria. In recent years, substantial progress has been made in understanding bacterial gene expression by sRNAs. However, recent findings that demonstrate that families of mRNAs show non-trivial sub-cellular distributions raise the question of how localization may affect the regulatory activity of sRNAs. Here we address this question within a simple mathematical model. We show that the non-uniform spatial distributions of mRNA can alter the threshold-linear response that characterizes sRNAs that act stoichiometrically, and modulate the hierarchy among targets co-regulated by the same sRNA. We also identify conditions where the sub-cellular organization of cofactors in the sRNA pathway can induce spatial heterogeneity on sRNA targets. Our results suggest that under certain conditions, interpretation and modeling of natural and synthetic gene regulatory circuits need to take into account the spatial organization of the transcripts of participating genes.

  12. Host factor I, Hfq, binds to Escherichia coli ompA mRNA in a growth rate-dependent fashion and regulates its stability

    DEFF Research Database (Denmark)

    Vytvytska, O; Jakobsen, J S; Balcunaite, G

    1998-01-01

    The stability of the ompA mRNA depends on the bacterial growth rate. The 5' untranslated region is the stability determinant of this transcript and the target of the endoribonuclease, RNase E, the key player of mRNA degradation. An RNA-binding protein with affinity for the 5' untranslated region......RNA. In hfq mutant cells with a deficient Hfq gene product, the RNA-binding activity is missing, and analysis of the ompA mRNA showed that the growth-rate dependence of degradation is lost. Furthermore, the half-life of the ompA mRNA is prolonged in the mutant cells, irrespective of growth rate. Hfq has...

  13. Association of PITX2 mRNA down-regulation in prostate cancer with promoter hypermethylation and poor prognosis.

    Science.gov (United States)

    Vinarskaja, Anna; Schulz, Wolfgang A; Ingenwerth, Marc; Hader, Christiane; Arsov, Christian

    2013-07-01

    Hypermethylation of the PITX2 (paired-like homeodomain transcription factor 2) gene promoter is strongly associated with recurrence after radical prostatectomy. We hypothesized that PITX2 hypermethylation leads to PITX2 silencing and that decreased PITX2 expression is likewise associated with poor prognosis in prostate cancers. Moreover, it is unknown so far how PITX2 hypermethylation relates to other molecular changes in prostate cancer, such as ERG oncogenic activation in about half of all cases. To investigate how PITX2 expression and methylation are related, whether biochemical recurrence after radical prostatectomy can be predicted by PITX2 mRNA levels, and how changes in PITX2 relate to ERG overexpression. We measured PITX2 and ERG expression in 45 cancerous and 13 benign tissues from patients undergoing radical prostatectomy (age range: 59-74 years). Methylation of the PITX2 gene was analyzed in an extended series of 93 cancers. Follow-up was performed for all patients for a 98-month median period. Additionally, expression and methylation changes of PITX2 were investigated in prostate carcinoma cell lines. Gene expression and methylation were determined by quantitative RT-PCR and methylation-specific PCR, respectively. Biochemical recurrence defined as a total PSA of >0.2 ng/ml on 2 consecutive tests was considered as the surrogate endpoint for survival analysis. PITX2 expression was significantly and strongly decreased in prostate cancer compared to benign tissues. Cases with decreased PITX2 experienced significantly earlier biochemical recurrences. PITX2 down-regulation was associated with PITX2 promoter hypermethylation in tumor samples and cell lines. PITX2 hypermethylation was more pronounced in cases with ERG overexpression. PITX2 down-regulation is associated with promoter hypermethylation and is a good predictor of clinical outcomes after radical prostatectomy. PITX2 methylation might be influenced by oncogenic ERG. Copyright © 2013 Elsevier Inc

  14. Regulation of the utilization of mRNA for eucaryotic elongation factor Tu in Friend erythroleukemia cells.

    Science.gov (United States)

    Rao, T R; Slobin, L I

    1987-02-01

    When Friend erythroleukemia cells were allowed to grow to stationary phase (2 X 10(6) to 3 X 10(6) cells per ml), approximately 60% of the mRNA for eucaryotic elongation factor Tu (eEF-Tu) sedimented at less than or equal to 80S, and most of the remaining factor mRNA was associated with small polysomes. Under the same growth conditions, greater than 90% of the mRNA for eucaryotic initiation factor 4A remained associated with polysomes. The association of eEF-Tu mRNA with polysomes changed dramatically when stationary-phase cells were treated with fresh medium. After 1 h in fresh medium, approximately 90% of eEF-Tu mRNA in Friend cells was found in heavy polysomes. Associated with the shift of eEF-Tu mRNA into heavy polysomes, we found at least a 2.6-fold increase in the synthesis of eEF-Tu in vivo as well as a remarkable 40% decrease in the total amount of eEF-Tu mRNA per cell. Our data raise the possibility that eEF-Tu mRNA that has accumulated in ribonucleoprotein particles in stationary-phase cells is degraded rather than reutilized for eEF-Tu synthesis.

  15. Mechanism of Regulation of bcl-2 mRNA by Nucleolin and A+U-rich Element-binding Factor 1 (AUF1)*

    Science.gov (United States)

    Ishimaru, Daniella; Zuraw, Lisa; Ramalingam, Sivakumar; Sengupta, Tapas K.; Bandyopadhyay, Sumita; Reuben, Adrian; Fernandes, Daniel J.; Spicer, Eleanor K.

    2010-01-01

    The antiapoptotic Bcl-2 protein is overexpressed in a variety of cancers, particularly leukemias. In some cell types this is the result of enhanced stability of bcl-2 mRNA, which is controlled by elements in its 3′-untranslated region. Nucleolin is one of the proteins that binds to bcl-2 mRNA, thereby increasing its half-life. Here, we examined the site on the bcl-2 3′-untranslated region that is bound by nucleolin as well as the protein binding domains important for bcl-2 mRNA recognition. RNase footprinting and RNA fragment binding assays demonstrated that nucleolin binds to a 40-nucleotide region at the 5′ end of the 136-nucleotide bcl-2 AU-rich element (AREbcl-2). The first two RNA binding domains of nucleolin were sufficient for high affinity binding to AREbcl-2. In RNA decay assays, AREbcl-2 transcripts were protected from exosomal decay by the addition of nucleolin. AUF1 has been shown to recruit the exosome to mRNAs. When MV-4-11 cell extracts were immunodepleted of AUF1, the rate of decay of AREbcl-2 transcripts was reduced, indicating that nucleolin and AUF1 have opposing roles in bcl-2 mRNA turnover. When the function of nucleolin in MV-4-11 cells was impaired by treatment with the nucleolin-targeting aptamer AS1411, association of AUF1 with bcl-2 mRNA was increased. This suggests that the degradation of bcl-2 mRNA induced by AS1411 results from both interference with nucleolin protection of bcl-2 mRNA and recruitment of the exosome by AUF1. Based on our findings, we propose a model that illustrates the opposing roles of nucleolin and AUF1 in regulating bcl-2 mRNA stability. PMID:20571027

  16. Mechanism of regulation of bcl-2 mRNA by nucleolin and A+U-rich element-binding factor 1 (AUF1).

    Science.gov (United States)

    Ishimaru, Daniella; Zuraw, Lisa; Ramalingam, Sivakumar; Sengupta, Tapas K; Bandyopadhyay, Sumita; Reuben, Adrian; Fernandes, Daniel J; Spicer, Eleanor K

    2010-08-27

    The antiapoptotic Bcl-2 protein is overexpressed in a variety of cancers, particularly leukemias. In some cell types this is the result of enhanced stability of bcl-2 mRNA, which is controlled by elements in its 3'-untranslated region. Nucleolin is one of the proteins that binds to bcl-2 mRNA, thereby increasing its half-life. Here, we examined the site on the bcl-2 3'-untranslated region that is bound by nucleolin as well as the protein binding domains important for bcl-2 mRNA recognition. RNase footprinting and RNA fragment binding assays demonstrated that nucleolin binds to a 40-nucleotide region at the 5' end of the 136-nucleotide bcl-2 AU-rich element (ARE(bcl-2)). The first two RNA binding domains of nucleolin were sufficient for high affinity binding to ARE(bcl-2). In RNA decay assays, ARE(bcl-2) transcripts were protected from exosomal decay by the addition of nucleolin. AUF1 has been shown to recruit the exosome to mRNAs. When MV-4-11 cell extracts were immunodepleted of AUF1, the rate of decay of ARE(bcl-2) transcripts was reduced, indicating that nucleolin and AUF1 have opposing roles in bcl-2 mRNA turnover. When the function of nucleolin in MV-4-11 cells was impaired by treatment with the nucleolin-targeting aptamer AS1411, association of AUF1 with bcl-2 mRNA was increased. This suggests that the degradation of bcl-2 mRNA induced by AS1411 results from both interference with nucleolin protection of bcl-2 mRNA and recruitment of the exosome by AUF1. Based on our findings, we propose a model that illustrates the opposing roles of nucleolin and AUF1 in regulating bcl-2 mRNA stability.

  17. MECHANISM AND REGULATION OF NONSENSE-MEDIATED MRNA DECAY (NMD, AN ESSENTIAL QUALITY CONTROL SYSTEM OF PLANTS

    Directory of Open Access Journals (Sweden)

    D. Silhavy

    2008-09-01

    Full Text Available In eukaryotic cell, various quality control mechanisms have evolved to ensure that only perfect mRNAs could be translated. Nonsense-mediated mRNA decay (NMD is a quality control system that identifies and eliminates mRNAs containing premature termination codons, thereby preventing the accumulation of potentially harmful truncated proteins. While NMD is well-characterized in yeast, in invertebrates and in mammals, plant NMD is poorly understood. In yeast and in invertebrates unusually long 3'untranslated regions (3'UTRs render an mRNA subject to NMD, while in mammals' 3'UTR located introns trigger NMD. UPF1, 2 and 3 are the key trans-acting NMD factors in yeast as well as in animals. However, in mammals, the core components of the Exon Junction Complex (Mago, Y14, eIF4A3 and MLN51 are also required for NMD. It was proposed that long 3’UTR-induced NMD is the ancient type and that it was changed to a more complex intron-based NMD in mammals. To better understand the evolution of eukaryotic NMD systems, we have studied the NMD machinery of plants, as plants are outgroup relative to fungi and animals. We have elaborated various transient assays to analyze plant NMD. Using these assays we defined the cis elements of plant NMD and characterized several trans-acting plant NMD factors. We demonstrated that two plant NMD pathways co-exist, one pathway, as yeast or invertebrate NMD systems, eliminates mRNAs with long 3'UTRs, while a distinct pathway, like mammalian NMD, degrades mRNAs harbouring 3'UTR-located introns. We showed that UPF1, UPF2, and SMG-7 are involved in both plant NMD pathways, whereas Mago and Y14 are required only for intron-based NMD. We also provide evidence that the molecular mechanism of long 3'UTR-based plant NMD resembles yeast NMD, while the intron-based NMD is similar to mammalian NMD. Moreover we have found that the SMG-7 component of plant NMD is targeted by NMD suggesting that plant NMD is autoregulated. We propose that in

  18. Klonierung der cDNA des Protein A Kinase-Adaptor-Proteins-2 und Untersuchungen zur Regulation seiner mRNA in humanen fötalen Osteoblasten

    OpenAIRE

    Reichel, Sonja

    2002-01-01

    In der vorliegenden Arbeit wurde erstmalig die vollständige cDNA-Sequenz des Proteinkinase A-Ankerproteins-2 (AKAP-2, 7,5kB) ermittelt. Zu diesem neuen humanen Gen sind bis dato außer einem unvollständigem Datenbankeintrag noch keine experimentellen Daten zur cDNA, mRNA oder zum Protein veröffentlicht. Mittels Northernblot wurde die Regulation der Expression der mRNA von AKAP-2 in humanem osteoblastären hFOB-Zellen sowie ihre Expression in unterschiedlichen Geweben untersucht: In hFOB-Zellen ...

  19. A tRNA-derived fragment competes with mRNA for ribosome binding and regulates translation during stress.

    Science.gov (United States)

    Gebetsberger, Jennifer; Wyss, Leander; Mleczko, Anna M; Reuther, Julia; Polacek, Norbert

    2017-10-03

    Posttranscriptional processing of RNA molecules is a common strategy to enlarge the structural and functional repertoire of RNomes observed in all 3 domains of life. Fragmentation of RNA molecules of basically all functional classes has been reported to yield smaller non-protein coding RNAs (ncRNAs) that typically possess different roles compared with their parental transcripts. Here we show that a valine tRNA-derived fragment (Val-tRF) that is produced under certain stress conditions in the halophilic archaeon Haloferax volcanii is capable of binding to the small ribosomal subunit. As a consequence of Val-tRF binding mRNA is displaced from the initiation complex which results in global translation attenuation in vivo and in vitro. The fact that the archaeal Val-tRF also inhibits eukaryal as well as bacterial protein biosynthesis implies a functionally conserved mode of action. While tRFs and tRNA halves have been amply identified in recent RNA-seq project, Val-tRF described herein represents one of the first functionally characterized tRNA processing products to date.

  20. Regulating Hemp and Cannabis-Based Products.

    Science.gov (United States)

    Bridges, Mindy; Hanson, Karmen

    2017-10-01

    (1)Different state agencies often regulate the hemp and cannabis industries separately. (2) Pharmaceutical products derived from cannabis are currently in clinical trials for approval in the U.S. and other countries. One product is already approved for use in 29 countries, but not in the U.S. (3) In August 2017, NCSL unanimously approved a policy position supporting federal legislation to define industrial hemp as a distinct agricultural crop (containing 1 percent or less of THC) and allow states to regulate commercial hemp farming.

  1. Single β-actin mRNA detection in neurons reveals a mechanism for regulating its translatability.

    Science.gov (United States)

    Buxbaum, Adina R; Wu, Bin; Singer, Robert H

    2014-01-24

    The physical manifestation of learning and memory formation in the brain can be expressed by strengthening or weakening of synaptic connections through morphological changes. Local actin remodeling underlies some forms of plasticity and may be facilitated by local β-actin synthesis, but dynamic information is lacking. In this work, we use single-molecule in situ hybridization to demonstrate that dendritic β-actin messenger RNA (mRNA) and ribosomes are in a masked, neuron-specific form. Chemically induced long-term potentiation prompts transient mRNA unmasking, which depends on factors active during synaptic activity. Ribosomes and single β-actin mRNA motility increase after stimulation, indicative of release from complexes. Hence, the single-molecule assays we developed allow for the quantification of activity-induced unmasking and availability for active translation. Further, our work demonstrates that β-actin mRNA and ribosomes are in a masked state that is alleviated by stimulation.

  2. Regulation and coupling of argECBH mRNA and enzyme synthesis in cell extracts of Escherichia coli.

    Science.gov (United States)

    Zidwick, M J; Keller, G; Rogers, P

    1984-08-01

    Cell extracts from Escherichia coli were used to study both transcription and coupled translation of the argECBH gene cluster. Argininosuccinase (the argH enzyme) and N-acetylornithinase (the argE enzyme) were synthesized for 90 to 120 min, and hybridizable argECBH mRNA was synthesized for 60 min after the addition of a lambda or phi 80 dargECBH DNA template. L-Arginine (2.5 mM) repressed synthesis by argR+ extracts of argECBH mRNA 2-, to 3-fold, argE enzyme 5- to 8-fold, and argH enzyme 20- to 60-fold. Repression was specific for L-arginine, and argR extracts were insensitive to added L-arginine. The argECBH mRNA made under conditions of restricted protein synthesis had reduced ability to function in the formation of the argE and argH enzymes and was found to be predominantly 6 to 8S in sucrose density gradients. When protein synthesis was allowed, the mRNA formed was functional, and large amounts of 14 to 23S argECBH mRNA appeared on sucrose gradients. An S-100 supernatant freed of ribosomes was capable of producing hybridizable arg mRNA, but significant functional message was only produced when ribosomes were present. When purified RNA polymerase was used, the formation of short 6 to 8S argECBH mRNA was dependent upon added rho protein. The data suggest that rho-dependent sites in the argECBH operon allow early termination of mRNA synthesis when transcription is not coupled to active enzyme synthesis.

  3. Regulation of ornithine decarboxylase during cell growth. Changes in the stability and translatability of the mRNA, and in the turnover of the protein.

    Science.gov (United States)

    Wallon, U M; Persson, L; Heby, O

    1995-05-10

    When Ehrlich ascites tumor cells were stimulated to grow, their ornithine decarboxylase (ODC) activity increased 20- to 30-fold. The increase in ODC mRNA content was one order of magnitude less during the corresponding period. Likewise, the subsequent changes in ODC activity failed to show proportionality to those of the ODC mRNA content. The changes in ODC activity were not attributable to changes in ODC turnover, even though the half-life of the enzyme decreased from 56 min during the period of increasing, to 36 min during the period of decreasing ODC activity. There was no evidence of an activation-inactivation-cycle for the enzyme. In view of these findings it appears that ODC mRNA alterations are amplified mainly at the translational level. The biphasic change in ODC mRNA content was partly attributable to a change in turnover of the message, as determined after inhibition of transcription with actinomycin D. Thus, the ODC mRNA half-life was estimated to decrease from 8.7 h during the period of increasing ODC activity to 4.0 h during the period of decreasing ODC activity. Despite the inhibition of transcription by actinomycin D, there was a marked superinduction of ODC activity. Our data demonstrate that the regulation of ODC expression is a complex phenomenon, involving controls at many levels.

  4. Stabilization of HIF-2α through redox regulation of mTORC2 activation and initiation of mRNA translation.

    Science.gov (United States)

    Nayak, B K; Feliers, D; Sudarshan, S; Friedrichs, W E; Day, R T; New, D D; Fitzgerald, J P; Eid, A; Denapoli, T; Parekh, D J; Gorin, Y; Block, K

    2013-06-27

    Hypoxia inducible factor-2α (HIF-2α) has a critical role in renal tumorigenesis. HIF-2α is stabilized in von Hippel-Lindau (VHL)-deficient renal cell carcinoma through mechanisms that require ongoing mRNA translation. Mammalian target of rapamycin (mTOR) functions in two distinct complexes: Raptor-associated mTORC1 and Rictor-associated mTORC2. Rictor-associated mTORC2 complex has been linked to maintaining HIF-2α protein in the absence of VHL; however, the mechanisms remain to be elucidated. Although Raptor-associated mTORC1 is a known key upstream regulator of mRNA translation, initiation and elongation, the role of mTORC2 in regulating mRNA translation is not clear. Complex assembly of the mRNA cap protein, eukaryotic translation initiation factor 4 (eIF4)E, with activators (eIF4 gamma (eIF4G)) and inhibitors (eIF4E-binding protein 1 (4E-BP1)) are rate-limiting determinants of mRNA translation. Our laboratory has previously demonstrated that reactive oxygen species, mediated by p22(phox)-based Nox oxidases, are enhanced in VHL-deficient cells and have a role in the activation of Akt on S473, a site phosphorylated by the mTORC2 complex. In this study, we examined the role of Rictor-dependent regulation of HIF-2α through eIF4E-dependent mRNA translation and examined the effects of p22(phox)-based Nox oxidases on TORC2 regulation. We demonstrate for the first time that mTORC2 complex stability and activation is redox sensitive, and further defined a novel role for p22(phox)-based Nox oxidases in eIF4E-dependent mRNA translation through mTORC2. Furthermore, we provide the first evidence that silencing of p22(phox) reduces HIF-2α-dependent gene targeting in vitro and tumor formation in vivo. The clinical relevance of these studies is demonstrated.

  5. Human gastrin mRNA expression up-regulated by Helicobacter pylori CagA through MEK/ERK and JAK2-signaling pathways in gastric cancer cells.

    Science.gov (United States)

    Zhou, Jianjiang; Xie, Yuan; Zhao, Yan; Wang, Shu; Li, Yu

    2011-10-01

    Helicobacter pylori-cytotoxin-associated protein A (CagA) and gastrin are believed to play an important role in gastric carcinogenesis, but their interaction has been incompletely clear. We constructed a eukaryotic expression vector pcDNA3.1/cagA and a luciferase reporter vector pGL/gastrin promoter, and then co-transfected them into gastric cancer AGS and SGC-7901 cells. The two kinds of gastric cancer cells were, respectively, infected with cagA-positive H. pylori NCTC11637, and then the gastrin promoter activity and gastrin mRNA level were detected with a Dual-Luciferase reporter assay system and quantitative reverse transcription polymerase chain reaction (RT-PCR), respectively. Next, after the MEK/ERK and JAK2-signaling pathway inhibitors, U0126 and AG490, were used to treat the two cell lines, or the ERK1 and JAK2 genes were knocked down by siRNAs (small interference RNAs) in the two cell lines, the gastrin promoter activity and gastrin mRNA level were observed again. The results indicated that CagA could activate the gastrin promoter and up-regulate gastrin mRNA expression in AGS and SGC-7901 cells, but these effects could be inhibited by the inhibitors U0126 and AG490, and the CagA-induced gastrin mRNA expression was down-regulated in the cells whose ERK1 or JAK2 gene was knocked down. Gastrin promoter may be the transcriptional target of CagA, and CagA activates the gastrin promoter to up-regulate gastrin mRNA expression through the MEK/ERK and JAK1-signaling pathway in gastric cancer cells.

  6. Identification of novel androgen-regulated pathways and mRNA isoforms through genome-wide exon-specific profiling of the LNCaP transcriptome.

    Directory of Open Access Journals (Sweden)

    Prabhakar Rajan

    Full Text Available Androgens drive the onset and progression of prostate cancer (PCa by modulating androgen receptor (AR transcriptional activity. Although several microarray-based studies have identified androgen-regulated genes, here we identify in-parallel global androgen-dependent changes in both gene and alternative mRNA isoform expression by exon-level analyses of the LNCaP transcriptome. While genome-wide gene expression changes correlated well with previously-published studies, we additionally uncovered a subset of 226 novel androgen-regulated genes. Gene expression pathway analysis of this subset revealed gene clusters associated with, and including the tyrosine kinase LYN, as well as components of the mTOR (mammalian target of rapamycin pathway, which is commonly dysregulated in cancer. We also identified 1279 putative androgen-regulated alternative events, of which 325 (∼25% mapped to known alternative splicing events or alternative first/last exons. We selected 30 androgen-dependent alternative events for RT-PCR validation, including mRNAs derived from genes encoding tumour suppressors and cell cycle regulators. Of seven positively-validating events (∼23%, five events involved transcripts derived from alternative promoters of known AR gene targets. In particular, we found a novel androgen-dependent mRNA isoform derived from an alternative internal promoter within the TSC2 tumour suppressor gene, which is predicted to encode a protein lacking an interaction domain required for mTOR inhibition. We confirmed that expression of this alternative TSC2 mRNA isoform was directly regulated by androgens, and chromatin immunoprecipitation indicated recruitment of AR to the alternative promoter region at early timepoints following androgen stimulation, which correlated with expression of alternative transcripts. Together, our data suggest that alternative mRNA isoform expression might mediate the cellular response to androgens, and may have roles in clinical

  7. Differential trypanosome surface coat regulation by a CCCH protein that co-associates with procyclin mRNA cis-elements.

    Directory of Open Access Journals (Sweden)

    Pegine Walrad

    2009-02-01

    Full Text Available The genome of Trypanosoma brucei is unusual in being regulated almost entirely at the post-transcriptional level. In terms of regulation, the best-studied genes are procyclins, which encode a family of major surface GPI-anchored glycoproteins (EP1, EP2, EP3, GPEET that show differential expression in the parasite's tsetse-fly vector. Although procyclin mRNA cis-regulatory sequences have provided the paradigm for post-transcriptional control in kinetoplastid parasites, trans-acting regulators of procyclin mRNAs are unidentified, despite intensive effort over 15 years. Here we identify the developmental regulator, TbZFP3, a CCCH-class predicted RNA binding protein, as an isoform-specific regulator of Procyclin surface coat expression in trypanosomes. We demonstrate (i that endogenous TbZFP3 shows sequence-specific co-precipitation of EP1 and GPEET, but not EP2 and EP3, procyclin mRNA isoforms, (ii that ectopic overexpression of TbZFP3 does not perturb the mRNA abundance of procyclin transcripts, but rather that (iii their protein expression is regulated in an isoform-specific manner, as evidenced by mass spectrometric analysis of the Procyclin expression signature in the transgenic cell lines. The TbZFP3 mRNA-protein complex (TbZFP3mRNP is identified as a trans-regulator of differential surface protein expression in trypanosomes. Moreover, its sequence-specific interactions with procyclin mRNAs are compatible with long-established predictions for Procyclin regulation. Combined with the known association of TbZFP3 with the translational apparatus, this study provides a long-sought missing link between surface protein cis-regulatory signals and the gene expression machinery in trypanosomes.

  8. 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

    Pathogen-associated molecular patterns (PAMPs) are conserved molecules of microorganisms inducing innate immune cells to secrete distinct patterns of cytokines. In veterinary species, due to a lack of specific antibodies, cytokines are often monitored as expressed mRNA only. This study investigated...... 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......-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...

  9. Increased peripheral nerve excitability and local NaV1.8 mRNA up-regulation in painful neuropathy

    Directory of Open Access Journals (Sweden)

    Matsuka Yoshizo

    2009-03-01

    Full Text Available Abstract Background Neuropathic pain caused by peripheral nerve injury is a chronic disorder that represents a significant clinical challenge because the pathological mechanisms have not been fully elucidated. Several studies have suggested the involvement of various sodium channels, including tetrodotoxin-resistant NaV1.8, in affected dorsal root ganglion (DRG neurons. We have hypothesized that altered local expression of NaV1.8 in the peripheral axons of DRG neurons could facilitate nociceptive signal generation and propagation after neuropathic injury. Results After unilateral sciatic nerve entrapment injury in rats, compound action potential amplitudes were increased in both myelinated and unmyelinated fibers of the ipsilateral sciatic nerve. Tetrodotoxin resistance of both fiber populations and sciatic nerve NaV1.8 immunoreactivity were also increased. Further analysis of NaV1.8 distribution revealed that immunoreactivity and mRNA levels were decreased and unaffected, respectively, in the ipsilateral L4 and L5 DRG; however sciatic nerve NaV1.8 mRNA showed nearly an 11-fold ipsilateral increase. Nav1.8 mRNA observed in the sciatic nerve was likely of axonal origin since it was not detected in non-neuronal cells cultured from nerve tissue. Absence of changes in NaV1.8 mRNA polyadenylation suggests that increased mRNA stability was not responsible for the selective peripheral mRNA increase. Furthermore, mRNA levels of NaV1.3, NaV1.5, NaV1.6, NaV1.7, and NaV1.9 were not significantly different between ipsilateral and contralateral nerves. We therefore propose that selective NaV1.8 mRNA axonal transport and local up-regulation could contribute to the hyperexcitability of peripheral nerves in some neuropathic pain states. Conclusion Cuff entrapment injury resulted in significantly elevated axonal excitability and increased NaV1.8 immunoreactivity in rat sciatic nerves. The concomitant axonal accumulation of NaV1.8 mRNA may play a role in the

  10. Regulation of intraluteal production of prostaglandins

    Directory of Open Access Journals (Sweden)

    Ottobre Joseph S

    2003-11-01

    Full Text Available Abstract There is clear evidence for intraluteal production of prostaglandins (PGs in numerous species and under a variety of experimental conditions. In general, secretion of PGs appears to be elevated in the early corpus luteum (CL and during the period of luteolysis. Regulation of intraluteal PG production is regulated by a variety of factors. An autoamplification pathway in which PGF-2alpha stimulates intraluteal production of PGF-2alpha has been identified in a number of species. The mechanisms underlying this autoamplification pathway appear to differ by species with expression of Cyclooxygenase-2 (Cox-2 and activity of phospholipase A2 acting as important physiological control points. In addition, a number of other responses that are induced by PGF-2alpha (decreased luteal progesterone, increased endothelin-1, increased cytokines also have been found to increase intraluteal PGF-2alpha production. Thus, regulation of intraluteal PG production may serve to initiate or amplify physiological signals to the CL and may be important in specific aspects of luteal physiology particularly during luteal regression.

  11. A 3' stem/loop structure of the Chlamydomonas chloroplast atpB gene regulates mRNA accumulation in vivo.

    Science.gov (United States)

    Stern, D B; Radwanski, E R; Kindle, K L

    1991-01-01

    The Chlamydomonas reinhardtii chloroplast atpB mRNA contains sequences at its 3' end that can form a complex stem/loop structure. Deletions of part or all of this sequence in transformed C. reinhardtii cells led to decreased atpB mRNA accumulation, whereas transcription rates were unaffected. The reduction of mRNA to 20% to 35% of wild-type levels in transformants without 3' stem/loops was correlated with the accumulation of atpB mRNA that was heterogeneous in size. These results indicated that RNA secondary structures function both in mRNA stabilization and in 3' end formation in C. reinhardtii chloroplasts. Furthermore, deletion of the stem/loop resulted in a decrease in the steady-state level of the ATPase beta-subunit to approximately 60% of wild-type levels, suggesting that translational and/or post-translational mechanisms may influence the steady-state level of the atpB gene product. PMID:1840911

  12. Tissue-specific expression and regulation by 1,25(OH)2D3 of chick protein kinase inhibitor (PKI) mRNA.

    Science.gov (United States)

    Marchetto, G S; Henry, H L

    1997-02-01

    The heat-stable protein kinase inhibitor (PKI) protein is a specific and potent competitive inhibitor of the catalytic subunit of cAMP-dependent protein kinase (PKA). Previously, it has been shown that vitamin D status affects chick kidney PKI activity: a 5- to 10-fold increase in PKI activity was observed in kidneys of chronically vitamin D-deficient chicks and treatment with 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) in cultured kidney cells resulted in a 95% decrease in PKI activity. The authors have recently cloned the cDNA for chick kidney PKI and have used the coding sequence to study the regulation of PKI mRNA. Northern analysis showed the expression of two PKI messages, which are 2.7 and 3.3 kb in size. These mRNAs are expressed in brain, muscle, testis, and kidney, but not in pancreas, liver, or intestine. PKI mRNA steady-state levels are downregulated by 47% in kidneys from vitamin D-replete chicks as compared to vitamin D-deficient chicks. PKI mRNA levels in brain, muscle, and testis are not affected by vitamin D status. Treatment of primary chick kidney cultures treated with 10(-7) M 1,25(OH)2D3 for 24h resulted in a 20-30% decrease in PKI mRNA. 1,25(OH)2D3 treatment does not affect the stability of PKI mRNA as determined by treatment of cell cultures with actinomycin D. This study shows that 1,25(OH)2D3 directly and tissue-specifically downregulates PKI mRNA in the chick kidney.

  13. p16(INK4A positively regulates p21(WAF1 expression by suppressing AUF1-dependent mRNA decay.

    Directory of Open Access Journals (Sweden)

    Huda H Al-Khalaf

    Full Text Available BACKGROUND: p16(INK4a and p21(WAF1 are two independent cyclin-dependent kinase inhibitors encoded by the CDKN2A and CDKN1A genes, respectively. p16(INK4a and p21(WAF1 are similarly involved in various anti-cancer processes, including the regulation of the critical G1 to S phase transition of the cell cycle, senescence and apoptosis. Therefore, we sought to elucidate the molecular mechanisms underlying the link between these two important tumor suppressor proteins. METHODOLOGY/PRINCIPAL FINDINGS: We have shown here that the p16(INK4a protein positively controls the expression of p21(WAF1 in both human and mouse cells. p16(INK4a stabilizes the CDKN1A mRNA through negative regulation of the mRNA decay-promoting AUF1 protein. Immunoprecipitation of AUF1-associated RNAs followed by quantitative RT-PCR indicated that endogenous AUF1 binds to the CDKN1A mRNA in a p16(INK4A-dependent manner. Furthermore, while AUF1 down-regulation increased the expression level of the CDKN1A mRNA, the concurrent knockdown of AUF1 and CDKN2A, using specific silencing RNAs, restored the normal expression of the gene. Moreover, we used EGFP reporter fused to the CDKN2A AU-rich element (ARE to demonstrate that p16(INK4A regulation of the CDKN1A mRNA is AUF1- and ARE-dependent. Furthermore, ectopic expression of p16(INK4A in p16(INK4A-deficient breast epithelial MCF-10A cells significantly increased the level of p21(WAF1, with no effect on cell proliferation. In addition, we have shown direct correlation between p16(INK4a and p21(WAF1 levels in various cancer cell lines. CONCLUSION/SIGNIFICANCE: These findings show that p16(INK4a stabilizes the CDKN1A mRNA in an AUF1-dependent manner, and further confirm the presence of a direct link between the 2 important cancer-related pathways, pRB/p16(INK4A and p14(ARF/p53/p21(WAF1.

  14. A comparative study of mRNA and protein expression of the autoimmune regulator gene (Aire) in embryonic and adult murine tissues.

    Science.gov (United States)

    Adamson, K A; Pearce, S H S; Lamb, J R; Seckl, J R; Howie, S E M

    2004-02-01

    Autoimmune polyendocrine syndrome type 1 (APS1) is a rare autosomal recessive human disorder caused by mutations in the autoimmune regulator gene (AIRE) and characterized by multiple autoimmune diseases. As reports of the tissue expression pattern of the murine Aire gene are discordant, a comprehensive survey of Aire expression was undertaken in adult and embryonic tissues at the mRNA and protein levels using real-time RT-PCR, in situ hybridization, and immunohistochemistry. In the adult, the highest Aire mRNA expression was in the thymus. All the other tissues investigated expressed Aire mRNA at low levels, but it was barely detectable in the adrenal gland. Aire protein expression was observed in the thymus, spleen, and lymph nodes. A common pattern was observed in other tissues, with staining in epithelial cells. An exception to this was the gut, where staining was seen in the mucin spaces. In embryonic tissue, Aire mRNA and protein expression was detected from E14.5 in the thymus. In the fetal liver, unlike the adult, staining was observed at E14.5 and decreased towards term. Thus, Aire is expressed in immunologically relevant tissues and in a restricted number of extra-immunological tissues in the adult. Furthermore, the presence of Aire protein is reported in extra-thymic tissues of the embryo. Copyright 2004 John Wiley & Sons, Ltd.

  15. siRNA down-regulation of FGA mRNA in HepG2 cells demonstrated that heterozygous abnormality of the Aα-chain gene does not affect the plasma fibrinogen level.

    Science.gov (United States)

    Takezawa, Yuka; Matsuda, Kazuyuki; Terasawa, Fumiko; Sugano, Mitsutoshi; Honda, Takayuki; Okumura, Nobuo

    2013-04-01

    We encountered two afibrinogenemia patients with homozygous and compound heterozygous FGA mutation. Of interest, the patients' parents, who are heterozygous, had normal levels of plasma fibrinogen; thus, we hypothesized that liver FGA mRNA levels were higher than those of FGB and/or FGG mRNA. To test the hypothesis, we quantitated mRNA levels of a normal liver and a human hepatocyte cell line, HepG2 cells, and performed siRNA-mediated down-regulation of the fibrinogen gene in HepG2 cells. mRNA levels were determined using real-time quantitative RT- PCR for three normal livers and HepG2 cells. Down-regulation of FGA, FGB, or FGG in HepG2 cells was performed by the addition of siRNA corresponding to each of the three genes, and the mRNA levels determined in the cells and the secreted fibrinogen concentration in media. The mRNA level of normal human liver was FGA=FGB>FGG and the FGG mRNA level was about 2-fold lower than the others, that of HepG2 cells was FGA>FGG>FGB and FGA mRNA was approximately 2- or 4-fold higher than FGG mRNA and FGB mRNA. When FGA, FGB, or FGG mRNA expression levels were down-regulated by nearby 50%, fibrinogen concentrations in media were 78%, 49%, or 57% of the control, respectively. Our results suggest that FGG mRNA levels limit fibrinogen expression in normal liver and HepG2 cells and that 50% reduction of FGA mRNA levels would not limit fibrinogen expression in normal liver and HepG2 cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Targeted mRNA Oxidation Regulates Sunflower Seed Dormancy Alleviation during Dry After-Ripening[C][W

    Science.gov (United States)

    Bazin, Jérémie; Langlade, Nicolas; Vincourt, Patrick; Arribat, Sandrine; Balzergue, Sandrine; El-Maarouf-Bouteau, Hayat; Bailly, Christophe

    2011-01-01

    After-ripening is the mechanism by which dormant seeds become nondormant during their dry storage after harvest. The absence of free water in mature seeds does not allow detectable metabolism; thus, the processes associated with dormancy release under these conditions are largely unknown. We show here that sunflower (Helianthus annuus) seed alleviation of dormancy during after-ripening is associated with mRNA oxidation and that this oxidation is prevented when seeds are maintained dormant. In vitro approaches demonstrate that mRNA oxidation results in artifacts in cDNA–amplified fragment length polymorphim analysis and alters protein translation. The oxidation of transcripts is not random but selective, and, using microarrays, we identified 24 stored mRNAs that became highly oxidized during after-ripening. Oxidized transcripts mainly correspond to genes involved in responses to stress and in cell signaling. Among them, protein phosphatase 2C PPH1, mitogen-activated protein kinase phosphatase 1, and phenyl ammonia lyase 1 were identified. We propose that targeted mRNA oxidation during dry after-ripening of dormant seeds could be a process that governs cell signaling toward germination in the early steps of seed imbibition. PMID:21642546

  17. The Crc global regulator binds to an unpaired A-rich motif at the Pseudomonas putida alkS mRNA coding sequence and inhibits translation initiation.

    Science.gov (United States)

    Moreno, Renata; Marzi, Stefano; Romby, Pascale; Rojo, Fernando

    2009-12-01

    Crc is a key global translational regulator in Pseudomonads that orchestrates the hierarchy of induction of several catabolic pathways for amino acids, sugars, hydrocarbons or aromatic compounds. In the presence of amino acids, which are preferred carbon sources, Crc inhibits translation of the Pseudomonas putida alkS and benR mRNAs, which code for transcriptional regulators of genes required to assimilate alkanes (hydrocarbons) and benzoate (an aromatic compound), respectively. Crc binds to the 5'-end of these mRNAs, but the sequence and/or structure recognized, and the way in which it inhibits translation, were unknown. We have determined the secondary structure of the alkS mRNA 5'-end through its sensitivity to several ribonucleases and chemical reagents. Footprinting and band-shift assays using variant alkS mRNAs have shown that Crc specifically binds to a short unpaired A-rich sequence located adjacent to the alkS AUG start codon. This interaction is stable enough to prevent formation of the translational initiation complex. A similar Crc-binding site was localized at benR mRNA, upstream of the Shine-Dalgarno sequence. This allowed predicting binding sites at other Crc-regulated genes, deriving a consensus sequence that will help to validate new Crc targets and to discriminate between direct and indirect effects of this regulator.

  18. Regulation of P450 oxidoreductase by gonadotropins in rat ovary and its effect on estrogen production

    Directory of Open Access Journals (Sweden)

    Uesaka Miki

    2008-12-01

    Full Text Available Abstract Background P450 oxidoreductase (POR catalyzes electron transfer to microsomal P450 enzymes. Its deficiency causes Antley-Bixler syndrome (ABS, and about half the patients with ABS have ambiguous genitalia and/or impaired steroidogenesis. POR mRNA expression is up-regulated when mesenchymal stem cells (MSCs differentiate into steroidogenic cells, suggesting that the regulation of POR gene expression is important for steroidogenesis. In this context we examined the regulation of POR expression in ovarian granulosa cells by gonadotropins, and its possible role in steroidogenesis. Methods Changes in gene expression in MSCs during differentiation into steroidogenic cells were examined by DNA microarray analysis. Changes in mRNA and protein expression of POR in the rat ovary or in granulosa cells induced by gonadotropin treatment were examined by reverse transcription-polymerase chain reaction and western blotting. Effects of transient expression of wild-type or mutant (R457H or V492E POR proteins on the production of estrone in COS-7 cells were examined in vitro. Effects of POR knockdown were also examined in estrogen producing cell-line, KGN cells. Results POR mRNA was induced in MSCs following transduction with the SF-1 retrovirus, and was further increased by cAMP treatment. Expression of POR mRNA, as well as Cyp19 mRNA, in the rat ovary were induced by equine chorionic gonadotropin and human chorionic gonadotropin. POR mRNA and protein were also induced by follicle stimulating hormone in primary cultured rat granulosa cells, and the induction pattern was similar to that for aromatase. Transient expression of POR in COS-7 cells, which expressed a constant amount of aromatase protein, greatly increased the rate of conversion of androstenedione to estrone, in a dose-dependent manner. The expression of mutant POR proteins (R457H or V492E, such as those found in ABS patients, had much less effect on aromatase activity than expression of wild

  19. An interplay between the p38 MAPK pathway and AUBPs regulates c-fos mRNA stability during mitogenic stimulation.

    Science.gov (United States)

    Degese, Maria Sol; Tanos, Tamara; Naipauer, Julian; Gingerich, Tim; Chiappe, Diego; Echeverria, Pablo; LaMarre, Jonathan; Gutkind, J Silvio; Coso, Omar A

    2015-04-01

    Mitogen-activated protein kinase (MAPK) pathways constitute key regulatory elements linking extracellular stimuli to nuclear gene expression. Immediate-early responsive genes (IEGs) of the activator protein 1 (AP-1) family, such as fos, achieve peak expression levels shortly after cells are stimulated with growth factors and sharply decrease thereafter. Several AU-rich binding proteins (AUBPs), including HuR (Hu-antigen R, Elav-like protein 1, ELAVL1) and KSRP (far upstream element-binding protein 2, KHSRP) bind to a fos AU-rich element (ARE) present in the 3'-UTR (untranslated region) of fos mRNA regulating its stability by a still poorly defined mechanism. We show in the present study that, whereas HuR binds and stabilizes transcribed reporter mRNAs bearing the fos 3'-UTR, KSRP counteracts this effect. Furthermore, we found that fos mRNA stability and HuR phosphorylation status are dependent on the activity of p38 MAPK in both epithelial cells and fibroblasts upon proliferative stimulation. Analysing PPI (protein-protein interaction) networks, we performed a thorough query of interacting proteins for p38 MAPKs, HuR and other AUBPs upon growth factor stimulation. This revealed novel HuR interactors including inhibitors of protein phosphatase 2 (PP2A) activity. Over-expression of two of these interactors, pp32 and APRIL (acidic leucine-rich nuclear phosphoprotein 32 family member B, ANP32B) and pharmacological inhibition of PP2A stabilized a fos reporter mRNA. Our results indicate that p38 MAPK regulates fos mRNA decay by affecting the state of phosphorylation of HuR while controlling yet to be fully elucidated PP regulatory networks.

  20. LMKB/MARF1 localizes to mRNA processing bodies, interacts with Ge-1, and regulates IFI44L gene expression.

    Directory of Open Access Journals (Sweden)

    Donald B Bloch

    Full Text Available The mRNA processing body (P-body is a cellular structure that regulates the stability of cytoplasmic mRNA. MARF1 is a murine oocyte RNA-binding protein that is associated with maintenance of mRNA homeostasis and genomic stability. In this study, autoantibodies were used to identify Limkain B (LMKB, the human orthologue of MARF1, as a P-body component. Indirect immunofluorescence demonstrated that Ge-1 (a central component of the mammalian core-decapping complex co-localized with LMKB in P-bodies. Two-hybrid and co-immunoprecipitation assays were used to demonstrate interaction between Ge-1 and LMKB. The C-terminal 120 amino acids of LMKB mediated interaction with Ge-1 and the N-terminal 1094 amino acids of Ge-1 were required for interaction with LMKB. LMKB is the first protein identified to date that interacts with this portion of Ge-1. LMKB was expressed in human B and T lymphocyte cell lines; depletion of LMKB increased expression of IFI44L, a gene that has been implicated in the cellular response to Type I interferons. The interaction between LMKB/MARF1, a protein that contains RNA-binding domains, and Ge-1, which interacts with core-decapping proteins, suggests that LMKB has a role in the regulation of mRNA stability. LMKB appears to have different functions in different cell types: maintenance of genomic stability in developing oocytes and possible dampening of the inflammatory response in B and T cells.

  1. Expression of inducible nitric oxide synthase mRNA and nitric oxide production during the development of liver abscess in hamster inoculated with Entamoeba histolytica.

    Science.gov (United States)

    Ramírez-Emiliano, Joel; González-Hernández, Angélica; Arias-Negrete, Sergio

    2005-06-01

    The present study analyzed iNOS and eNOS mRNA expression and NO production during development of hepatic abscess caused by Entamoeba histolytica trophozoites. One 374-bp sequence, which displayed 88% identity to mammalian iNOS protein, was isolated from LPS-stimulated peritoneal hamster macrophages. A separate 365-bp cDNA sequence showed 99% identity with eNOS protein. iNOS mRNA was detected in hamsters during formation of amoebic liver abscesses, but not in control hamsters. eNOS mRNA expression was not modified. Serum nitrite concentration in hamsters infected with E. histolytica was 33 +/- 6 microM, in control hamsters was 20 +/- 3 microM. The study shows that iNOS mRNA expression and NO production are induced by E. histolytica trophozoites during amoebic liver abscess formation. However, in spite of iNOS mRNA expression and NO production, E. histolytica trophozoites induced liver abscess formation in hamster.

  2. Crystal Structure of the N-Terminal RNA Recognition Motif of mRNA Decay Regulator AUF1

    Directory of Open Access Journals (Sweden)

    Young Jun Choi

    2016-01-01

    Full Text Available AU-rich element binding/degradation factor 1 (AUF1 plays a role in destabilizing mRNAs by forming complexes with AU-rich elements (ARE in the 3′-untranslated regions. Multiple AUF1-ARE complexes regulate the translation of encoded products related to the cell cycle, apoptosis, and inflammation. AUF1 contains two tandem RNA recognition motifs (RRM and a Gln- (Q- rich domain in their C-terminal region. To observe how the two RRMs are involved in recognizing ARE, we obtained the AUF1-p37 protein covering the two RRMs. However, only N-terminal RRM (RRM1 was crystallized and its structure was determined at 1.7 Å resolution. It appears that the RRM1 and RRM2 separated before crystallization. To demonstrate which factors affect the separate RRM1-2, we performed limited proteolysis using trypsin. The results indicated that the intact proteins were cleaved by unknown proteases that were associated with them prior to crystallization. In comparison with each of the monomers, the conformations of the β2-β3 loops were highly variable. Furthermore, a comparison with the RRM1-2 structures of HuR and hnRNP A1 revealed that a dimer of RRM1 could be one of the possible conformations of RRM1-2. Our data may provide a guidance for further structural investigations of AUF1 tandem RRM repeat and its mode of ARE binding.

  3. Oxygen regulation of uricase and sucrose synthase synthesis in soybean callus tissue is exerted at the mRNA level

    DEFF Research Database (Denmark)

    Xue, Z T; Larsen, K; Jochimsen, B U

    1991-01-01

    The effect of lowering oxygen concentration on the expression of nodulin genes in soybean callus tissue devoid of the microsymbiont has been examined. Poly(A)+ RNA was isolated from tissue cultivated in 4% oxygen and in normal atmosphere. Quantitative mRNA hybridization experiments using nodule-s...... was about 5-fold at 4% oxygen. No expression at atmospheric oxygen or in response to low oxygen was observed when using cDNA probes for other nodulin genes such as leghemoglobin c3, nodulin-22 and nodulin-44. Udgivelsesdato: 1991-May...

  4. Multiple transcripts regulate glucose-triggered mRNA decay of the lactate transporter JEN1 from Saccharomyces cerevisiae

    OpenAIRE

    Andrade, Raquel P.; Kötter, Peter; Entian, Karl-Dieter; Casal, Margarida

    2005-01-01

    The Saccharomyces cerevisiae JEN1 gene encoding the lactate transporter undergoes strong catabolic repression at both transcriptional and post-transcriptional levels. JEN1 mRNA decay is greatly accelerated upon the addition of a pulse of glucose, fructose or mannose to induced cell cultures. Mapping of the 5´UTRs and 3´UTRs of JEN1 transcripts revealed multiple transcription start-sites located at position -51, +391 or +972, depending on the cell culture conditions. The presence of t...

  5. Genomic analysis suggests that mRNA destabilization by the microprocessor is specialized for the auto-regulation of Dgcr8.

    Directory of Open Access Journals (Sweden)

    Archana Shenoy

    2009-09-01

    Full Text Available The Microprocessor, containing the RNA binding protein Dgcr8 and RNase III enzyme Drosha, is responsible for processing primary microRNAs to precursor microRNAs. The Microprocessor regulates its own levels by cleaving hairpins in the 5'UTR and coding region of the Dgcr8 mRNA, thereby destabilizing the mature transcript.To determine whether the Microprocessor has a broader role in directly regulating other coding mRNA levels, we integrated results from expression profiling and ultra high-throughput deep sequencing of small RNAs. Expression analysis of mRNAs in wild-type, Dgcr8 knockout, and Dicer knockout mouse embryonic stem (ES cells uncovered mRNAs that were specifically upregulated in the Dgcr8 null background. A number of these transcripts had evolutionarily conserved predicted hairpin targets for the Microprocessor. However, analysis of deep sequencing data of 18 to 200nt small RNAs in mouse ES, HeLa, and HepG2 indicates that exonic sequence reads that map in a pattern consistent with Microprocessor activity are unique to Dgcr8.We conclude that the Microprocessor's role in directly destabilizing coding mRNAs is likely specifically targeted to Dgcr8 itself, suggesting a specialized cellular mechanism for gene auto-regulation.

  6. S-Adenosylmethionine Synthesis Is Regulated by Selective N6-Adenosine Methylation and mRNA Degradation Involving METTL16 and YTHDC1

    Directory of Open Access Journals (Sweden)

    Hiroki Shima

    2017-12-01

    Full Text Available S-adenosylmethionine (SAM is an important metabolite as a methyl-group donor in DNA and histone methylation, tuning regulation of gene expression. Appropriate intracellular SAM levels must be maintained, because methyltransferase reaction rates can be limited by SAM availability. In response to SAM depletion, MAT2A, which encodes a ubiquitous mammalian methionine adenosyltransferase isozyme, was upregulated through mRNA stabilization. SAM-depletion reduced N6-methyladenosine (m6A in the 3′ UTR of MAT2A. In vitro reactions using recombinant METTL16 revealed multiple, conserved methylation targets in the 3′ UTR. Knockdown of METTL16 and the m6A reader YTHDC1 abolished SAM-responsive regulation of MAT2A. Mutations of the target adenine sites of METTL16 within the 3′ UTR revealed that these m6As were redundantly required for regulation. MAT2A mRNA methylation by METTL16 is read by YTHDC1, and we suggest that this allows cells to monitor and maintain intracellular SAM levels.

  7. The 5' leader of the mRNA encoding the mouse neurotrophin receptor TrkB contains two internal ribosomal entry sites that are differentially regulated.

    Directory of Open Access Journals (Sweden)

    Stephanie L Timmerman

    2007-09-01

    Full Text Available A single internal ribosomal entry site (IRES in conjunction with IRES transactivating factors (ITAFs is sufficient to recruit the translational machinery to a eukaryotic mRNA independent of the cap structure. However, we demonstrate that the mouse TrkB mRNA contains two independent IRESes. The mouse TrkB mRNA consists of one of two 5' leaders (1428 nt and 448 nt, both of which include the common 3' exon (Ex2, 344 nt. Dicistronic RNA transfections and in vitro translation of monocistronic RNA demonstrated that both full-length 5' leaders, as well as Ex2, exhibit IRES activity indicating the IRES is located within Ex2. Additional analysis of the upstream sequences demonstrated that the first 260 nt of exon 1 (Ex1a also contains an IRES. Dicistronic RNA transfections into SH-SY5Y cells showed the Ex1a IRES is constitutively active. However, the Ex2 IRES is only active in response to retinoic acid induced neural differentiation, a state which correlates with the synthesis of the ITAF polypyrimidine tract binding protein (PTB1. Correspondingly, addition or knock-down of PTB1 altered Ex2, but not Ex1a IRES activity in vitro and ex vivo, respectively. These results demonstrate that the two functionally independent IRESes within the mouse TrkB 5' leader are differentially regulated, in part by PTB1.

  8. Effects of exogenous plant growth regulator abscisic acid-induced resistance in rice on the expression of vitellogenin mRNA in Nilaparvata lugens (Hemiptera: Delphacidae) adult females.

    Science.gov (United States)

    Liu, Jing-Lan; Chen, Xiao; Zhang, Hong-Mei; Yang, Xia; Wong, Andrew

    2014-01-01

    Recent study showed that exogenous abscisic acid (ABA) acts as a regulator of plant resistance. This study investigated average injury scale and callose contents of rice, and vitellogenin (Nlvg) mRNA expression in Nilaparvata lugens (Stål) (Hemiptera: Delphacidae) adult females after third instar nymphs fed on exogenous ABA-treated susceptible [Taichung Native one (TN1)] and moderately resistant (IR42) rice cultivars. The results showed that exogenous ABA significantly decreased average injury scale of rice and Nlvg mRNA expression in N. lugens adults compared with the control (without ABA spraying). Nlvg mRNA expression in N. lugens adults decreased significantly after third instar nymphs fed on ABA-treated (5, 20, and 40 mg/liter) TN1 for 1 and 2 d, and for IR42, after fed on ABA-treated (20 and 40 mg/liter) rice plants for 1 d and after fed on ABA-treated (5, 20, and 40 mg/liter) rice for 2 d decreased significantly. The callose contents showed no significant change for TN1, while for IR42, significantly increased in roots and sheathes after N. lugens infestation under ABA treatments (20 and 40 mg/liter) compared with the control. The decrease of Nlvg mRNA expression may be partially attributed to the increase of callose content of plants. The results provide a profile for concerning the effects of ABA-induced rice plants' defenses on phloem-feeding insects. © The Author 2014. Published by Oxford University Press on behalf of the Entomological Society of America.

  9. Interleukin-8 and vascular endothelial growth factor mRNA and protein levels are down-regulated in ovarian carcinoma cells in serous effusions.

    Science.gov (United States)

    Davidson, Ben; Reich, Reuven; Kopolovic, Juri; Berner, Aasmund; Nesland, Jahn M; Kristensen, Gunnar B; Tropé, Claes G; Bryne, Magne; Risberg, Bjørn; van de Putte, Gregg; Goldberg, Iris

    2002-01-01

    Angiogenic factors are involved in tumor growth and spread. The aim of this study was to evaluate the expression of angiogenesis-related genes in malignant serous effusions of patients with advanced-stage (FIGO stage III and IV) ovarian carcinoma. In addition, to compare the results for carcinoma cells in effusions with corresponding primary tumors and metastatic lesions, and analyze their prognostic role. Sections from 66 effusions and 90 primary and metastatic lesions from 62 ovarian and primary peritoneal carcinoma patients, were evaluated for expression of basic fibroblast factor (bFGF), interleukin-8 (IL-8), and vascular endothelial growth factor (VEGF) using mRNA in situ hybridization (ISH). Protein expression was evaluated in a subset of specimens using immunohistochemistry (IHC). ISH results were correlated with clinical parameters. In both effusions and solid tumors, bFGF mRNA was the most commonly expressed factor (93% of effusions and 95% of solid tumors) followed by IL-8, while VEGF was expressed in a minority of the specimens (P 0.05). Peritoneal and pleural effusions showed similar expression patterns. In conclusion, bFGF is the major angiogenic factor expressed in ovarian carcinoma at the mRNA level. It is highly expressed in both solid tumors and serous effusions, while IL-8 and VEGF are down regulated in carcinoma cells in effusions, possibly due to the lack of interaction with stromal cells. mRNA expression of VEGF, bFGF, and IL-8 does not appear to be a predictor of disease outcome in advanced-stage ovarian carcinoma. Carcinoma cells in pleural and peritoneal effusions show a similar metastatic expression profile, in agreement with our previous findings, supporting the true metastatic nature of ovarian carcinoma cells in ascites.

  10. 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

  11. NITRIC OXIDE PRODUCTION AND iNOS mRNA EXPRESSION IN IFN-8-STIMULATED CHICKEN MACROPHAGES TRANSFECTED WITH iNOS siRNAs

    Science.gov (United States)

    Utilizing RNA interference technology with siRNA in the HD-11 macrophage cell line, we determined how the knock-down of the iNOS (inducible nitric oxide synthase) gene affected IFN-' induced macrophage production of nitric oxide (NO) and mRNA expression of genes involved in this biological pathway i...

  12. Modeling rotavirus-like particles production in a baculovirus expression vector system: Infection kinetics, baculovirus DNA replication, mRNA synthesis and protein production.

    Science.gov (United States)

    Roldão, António; Vieira, Helena L A; Charpilienne, Annie; Poncet, Didier; Roy, Polly; Carrondo, Manuel J T; Alves, Paula M; Oliveira, R

    2007-03-10

    Rotavirus is the most common cause of severe diarrhoea in children worldwide, responsible for more than half a million deaths in children per year. Rotavirus-like particles (Rota VLPs) are excellent vaccine candidates against rotavirus infection, since they are non-infectious, highly immunogenic, amenable to large-scale production and safer to produce than those based on attenuated viruses. This work focuses on the analysis and modeling of the major events taking place inside Spodoptera frugiperda (Sf-9) cells infected by recombinant baculovirus that may be critical for the expression of rotavirus viral proteins (VPs). For model validation, experiments were performed adopting either a co-infection strategy, using three monocistronic recombinant baculovirus each one coding for viral proteins VP(2), VP(6) and VP(7), or single-infection strategies using a multigene baculovirus coding for the three proteins of interest. A characteristic viral DNA (vDNA) replication rate of 0.19+/-0.01 h(-1) was obtained irrespective of the monocistronic or multigene vector employed, and synthesis of progeny virus was found to be negligible in comparison to intracellular vDNA concentrations. The timeframe for vDNA, mRNA and VP synthesis tends to decrease with increasing multiplicity of infection (MOI) due to the metabolic burden effect. The protein synthesis rates could be ranked according to the gene size in the multigene experiments but not in the co-infection experiments. The model exhibits acceptable prediction power of the dynamics of intracellular vDNA replication, mRNA synthesis and VP production for the three proteins involved. This model is intended to be the basis for future Rota VLPs process optimisation and also a means to evaluating different baculovirus constructs for Rota VLPs production.

  13. Complex control of GABA(A receptor subunit mRNA expression: variation, covariation, and genetic regulation.

    Directory of Open Access Journals (Sweden)

    Megan K Mulligan

    Full Text Available GABA type-A receptors are essential for fast inhibitory neurotransmission and are critical in brain function. Surprisingly, expression of receptor subunits is highly variable among individuals, but the cause and impact of this fluctuation remains unknown. We have studied sources of variation for all 19 receptor subunits using massive expression data sets collected across multiple brain regions and platforms in mice and humans. Expression of Gabra1, Gabra2, Gabrb2, Gabrb3, and Gabrg2 is highly variable and heritable among the large cohort of BXD strains derived from crosses of fully sequenced parents--C57BL/6J and DBA/2J. Genetic control of these subunits is complex and highly dependent on tissue and mRNA region. Remarkably, this high variation is generally not linked to phenotypic differences. The single exception is Gabrb3, a locus that is linked to anxiety. We identified upstream genetic loci that influence subunit expression, including three unlinked regions of chromosome 5 that modulate the expression of nine subunits in hippocampus, and that are also associated with multiple phenotypes. Candidate genes within these loci include, Naaa, Nos1, and Zkscan1. We confirmed a high level of coexpression for subunits comprising the major channel--Gabra1, Gabrb2, and Gabrg2--and identified conserved members of this expression network in mice and humans. Gucy1a3, Gucy1b3, and Lis1 are novel and conserved associates of multiple subunits that are involved in inhibitory signaling. Finally, proximal and distal regions of the 3' UTRs of single subunits have remarkably independent expression patterns in both species. However, corresponding regions of different subunits often show congruent genetic control and coexpression (proximal-to-proximal or distal-to-distal, even in the absence of sequence homology. Our findings identify novel sources of variation that modulate subunit expression and highlight the extraordinary capacity of biological networks to buffer

  14. Bixin regulates mRNA expression involved in adipogenesis and enhances insulin sensitivity in 3T3-L1 adipocytes through PPARgamma activation.

    Science.gov (United States)

    Takahashi, Nobuyuki; Goto, Tsuyoshi; Taimatsu, Aki; Egawa, Kahori; Katoh, Sota; Kusudo, Tatsuya; Sakamoto, Tomoya; Ohyane, Chie; Lee, Joo-Young; Kim, Young-Il; Uemura, Taku; Hirai, Shizuka; Kawada, Teruo

    2009-12-25

    Insulin resistance is partly due to suppression of insulin-induced glucose uptake into adipocytes. The uptake is dependent on adipocyte differentiation, which is controlled at mRNA transcription level. The peroxisome proliferator-activated receptor (PPAR), a ligand-regulated nuclear receptor, is involved in the differentiation. Many food-derived compounds serve as ligands to activate or inactivate PPAR. In this study, we demonstrated that bixin and norbixin (annatto extracts) activate PPARgamma by luciferase reporter assay using GAL4-PPAR chimera proteins. To examine the effects of bixin on adipocytes, 3T3-L1 adipocytes were treated with bixin or norbixin. The treatment induced mRNA expression of PPARgamma target genes such as adipocyte-specific fatty acid-binding protein (aP2), lipoprotein lipase (LPL), and adiponectin in differentiated 3T3-L1 adipocytes and enhanced insulin-dependent glucose uptake. The observations indicate that bixin acts as an agonist of PPARgamma and enhances insulin sensitivity in 3T3-L1 adipocytes, suggesting that bixin is a valuable food-derived compound as a PPAR ligand to regulate lipid metabolism and to ameliorate metabolic syndrome.

  15. Bixin regulates mRNA expression involved in adipogenesis and enhances insulin sensitivity in 3T3-L1 adipocytes through PPAR{gamma} activation

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, Nobuyuki; Goto, Tsuyoshi; Taimatsu, Aki; Egawa, Kahori; Katoh, Sota; Kusudo, Tatsuya; Sakamoto, Tomoya; Ohyane, Chie; Lee, Joo-Young; Kim, Young-il; Uemura, Taku; Hirai, Shizuka [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji 611-0011 (Japan); Kawada, Teruo, E-mail: fat@kais.kyoto-u.ac.jp [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji 611-0011 (Japan)

    2009-12-25

    Insulin resistance is partly due to suppression of insulin-induced glucose uptake into adipocytes. The uptake is dependent on adipocyte differentiation, which is controlled at mRNA transcription level. The peroxisome proliferator-activated receptor (PPAR), a ligand-regulated nuclear receptor, is involved in the differentiation. Many food-derived compounds serve as ligands to activate or inactivate PPAR. In this study, we demonstrated that bixin and norbixin (annatto extracts) activate PPAR{gamma} by luciferase reporter assay using GAL4-PPAR chimera proteins. To examine the effects of bixin on adipocytes, 3T3-L1 adipocytes were treated with bixin or norbixin. The treatment induced mRNA expression of PPAR{gamma} target genes such as adipocyte-specific fatty acid-binding protein (aP2), lipoprotein lipase (LPL), and adiponectin in differentiated 3T3-L1 adipocytes and enhanced insulin-dependent glucose uptake. The observations indicate that bixin acts as an agonist of PPAR{gamma} and enhances insulin sensitivity in 3T3-L1 adipocytes, suggesting that bixin is a valuable food-derived compound as a PPAR ligand to regulate lipid metabolism and to ameliorate metabolic syndrome.

  16. Nerve growth factor regulation and production by macrophages in osteoarthritic synovium.

    Science.gov (United States)

    Takano, S; Uchida, K; Inoue, G; Miyagi, M; Aikawa, J; Iwase, D; Iwabuchi, K; Matsumoto, T; Satoh, M; Mukai, M; Minatani, A; Takaso, M

    2017-11-01

    Nerve growth factor (NGF) functions to modulate osteoarthritis (OA)-associated pain. Although recent studies suggest that tumour necrosis factor (TNF)-α and interleukin (IL)-1β mediate NGF activity in human synovial fibroblasts, the regulation of NGF expression in human synovial macrophages remains unclear. Here, we examined the role of macrophages in the production and regulation of synovial (SYN) NGF in osteoarthritic knee joints by examining the mRNA expression of TNF-α and IL-1β in freshly isolated CD14-positive (macrophage-rich fraction) and CD14-negative cells (fibroblast-rich fraction) in synovial tissue from OA patients by quantitative polymerase chain reaction. We also examined the effects of IL-1β and TNF-α on NGF mRNA expression in cultured CD14-positive (macrophage-rich fraction) and CD14-negative cells (fibroblast-rich fraction). In addition, to examine the contribution of macrophages to NGF, TNF-α and IL-1β expression, we injected clodronate liposomes systemically into STR/Ort mice, an osteoarthritis animal model, to deplete macrophages. TNF-α and IL-1β mRNA levels in CD14-positive cells from the SYN of OA patients was significantly higher than that in CD14-negative cells, while NGF expression did not differ markedly between the two cell fractions. In addition, treatment of human cultured CD14-positive and -negative cells with IL-1β and TNF-α enhanced NGF mRNA and protein levels. Expression of NGF, IL-1β and TNF-α was also reduced significantly in STR/Ort mice upon macrophage depletion. These findings suggest that IL-1β and TNF-α regulate NGF expression and production in synovial macrophages and fibroblasts in osteoarthritic joints. © 2017 British Society for Immunology.

  17. Orphan drug product regulation--United States.

    Science.gov (United States)

    Haffner, M E

    2002-02-01

    The legislative history of the United States Orphan Drug Act began with rare, unanimous approval by the United States Congress. The Act, mid consequently the Regulations, have evolved since then. The two-stage process of Orphan Drug designation and approval is outlined, as well as the incentives that are offered to commercial companies for their implementation. Orphan Drugs are likely to be over-represented among drugs used under "Treatment" INDs. For patent- and "drug-difference" reasons, the benefits under the Orphan Drug Act are especially valuable to those who develop biologics. By any measure, this legislation, which requires only voluntary participation, has been a success; because the human genome is likely to lead to more biologicals than orthodox drugs, this success is likely to continue into the future. But even so, the 18-year experience with Orphan Drugs in the United States has led to some 225 Orphan Product approvals that benefit many millions of patients.

  18. Eukaryotic initiation factor 3 (eIF3) and 5’ mRNA leader sequences as agents of translational regulation in Arabidopsis. Final report

    Energy Technology Data Exchange (ETDEWEB)

    von Arnim, Albrecht G. [Univ. of Tennessee, Knoxville, TN (United States)

    2015-02-04

    Protein synthesis, or translation, consumes a sizable fraction of the cell’s energy budget, estimated at 5% and up to 50% in differentiated and growing cells, respectively. Plants also invest significant energy and biomass to construct and maintain the translation apparatus. Translation is regulated by a variety of external stimuli. Compared to transcriptional control, attributes of translational control include reduced sensitivity to stochastic fluctuation, a finer gauge of control, and more rapid responsiveness to environmental stimuli. Yet, our murky understanding of translational control allows few generalizations. Consequently, translational regulation is underutilized in the context of transgene regulation, although synthetic biologists are now beginning to appropriate RNA-level gene regulation into their regulatory circuits. We also know little about how translational control contributes to the diversity of plant form and function. This project explored how an emerging regulatory mRNA sequence element, upstream open reading frames (uORFs), is integrated with the general translation initiation machinery to permit translational regulation on specific mRNAs.

  19. Effects of disruption of the nucleotide pattern in CRID element and Kozak sequence of interferon β on mRNA stability and protein production.

    Science.gov (United States)

    Kay, Maryam; Hojati, Zohreh; Heidari, Maryam; Bazi, Zahra; Korbekandi, Hassan

    2015-01-01

    Interferon β (IFNβ) is the most important drug that has been used frequently for multiple sclerosis treatment. This study has tried to improve the IFNβ production by introducing mutations in the coding region of IFNβ, while its amino acid sequence is intact. Two recombinant vectors IFNβ(K) and IFNβ(K+CRID )were designed by site-directed mutagenesis. The IFNβ(K) and IFNβ(K+CRID) have two substitutions in Kozak sequence and four substitutions in CRID sequence, respectively. The Chinese hamster ovary (CHO) cell codon usage optimization was also performed for both of them. They were transiently transfected to CHO-dhfr(-) cell line using Lipofectamine kit (Invitrogen, Grand Island, NY). The amount of mRNA and protein was determined by real time PCR and ELISA. The results of this study indicate that the amount of IFNβ protein produced by CHO cells containing IFNβ(K) has been elevated up to 3.5-fold. On the other hand, enormous amounts of IFNβ mRNA and protein were produced by cells containing IFNβ(K+CRID) construct; more than 4.6-fold and 6-fold, respectively. It could be concluded that disruption of AT pattern in CRID element increase RNA and protein production, improve IFNβ mRNA stability and, may also enhance mRNA half-life. In a similar way, more proteins are produced by modification of Kozak sequence.

  20. TREM-1 SNP rs2234246 regulates TREM-1 protein and mRNA levels and is associated with plasma levels of L-selectin.

    Directory of Open Access Journals (Sweden)

    Alex-Ander Aldasoro Arguinano

    Full Text Available High levels of TREM-1 are associated with cardiovascular and inflammatory diseases risks and the most recent studies have showed that TREM-1 deletion or blockade is associated with up to 60% reduction of the development of atherosclerosis. So far, it is unknown whether the levels of TREM-1 protein are genetically regulated. Moreover, TREM family receptors have been suggested to regulate the cellular adhesion process. The goal of this study was to investigate whether polymorphisms within TREM-1 are regulating the variants of serum TREM-1 levels and the expression levels of their mRNA. Furthermore, we aimed to point out associations between polymorphisms on TREM-1 and blood levels of selectins. Among the 10 SNPs studied, the minor allele T of rs2234246, was associated with increased sTREM-1 in the discovery population (p-value = 0.003, explaining 33% of its variance, and with increased levels of mRNA (p-value = 0.007. The same allele was associated with increased soluble L-selectin levels (p-value = 0.011. The higher levels of sTREM-1 and L-selectin were confirmed in the replication population (p-value = 0.0007 and p-value = 0.018 respectively. We demonstrated for the first time one SNP on TREM-1, affecting its expression levels. These novel results, support the hypothesis that TREM-1 affects monocytes extravasation and accumulation processes leading to atherogenesis and atherosclerotic plaque progression, possibly through increased inflammation and subsequent higher expression of sL-selectin.

  1. Regulation of the epithelial Ca2+ channels in small intestine as studied by quantitative mRNA detection.

    NARCIS (Netherlands)

    Abel, M. van; Hoenderop, J.G.J.; Kemp, J.W.C.M. van der; Leeuwen, J.P.P.M. van; Bindels, R.J.M.

    2003-01-01

    The epithelial Ca2+ channels TRPV5 and TRPV6 are localized to the brush border membrane of intestinal cells and constitute the postulated rate-limiting entry step of active Ca2+ absorption. The aim of the present study was to investigate the hormonal regulation of these channels. To this end, the

  2. Inferring microRNA regulation of mRNA with partially ordered samples of paired expression data and exogenous prediction algorithms.

    Directory of Open Access Journals (Sweden)

    Brian Godsey

    Full Text Available MicroRNAs (miRs are known to play an important role in mRNA regulation, often by binding to complementary sequences in "target" mRNAs. Recently, several methods have been developed by which existing sequence-based target predictions can be combined with miR and mRNA expression data to infer true miR-mRNA targeting relationships. It has been shown that the combination of these two approaches gives more reliable results than either by itself. While a few such algorithms give excellent results, none fully addresses expression data sets with a natural ordering of the samples. If the samples in an experiment can be ordered or partially ordered by their expected similarity to one another, such as for time-series or studies of development processes, stages, or types, (e.g. cell type, disease, growth, aging, there are unique opportunities to infer miR-mRNA interactions that may be specific to the underlying processes, and existing methods do not exploit this. We propose an algorithm which specifically addresses [partially] ordered expression data and takes advantage of sample similarities based on the ordering structure. This is done within a Bayesian framework which specifies posterior distributions and therefore statistical significance for each model parameter and latent variable. We apply our model to a previously published expression data set of paired miR and mRNA arrays in five partially ordered conditions, with biological replicates, related to multiple myeloma, and we show how considering potential orderings can improve the inference of miR-mRNA interactions, as measured by existing knowledge about the involved transcripts.

  3. LncRNA lnc-RI regulates homologous recombination repair of DNA double-strand breaks by stabilizing RAD51 mRNA as a competitive endogenous RNA.

    Science.gov (United States)

    Shen, Liping; Wang, Qi; Liu, Ruixue; Chen, Zhongmin; Zhang, Xueqing; Zhou, Pingkun; Wang, Zhidong

    2017-12-04

    DNA double-strand break (DSB) repair is critical for the maintenance of genome stability. The current models of the mechanism of DSB repair are based on studies of DNA repair proteins. Long non-coding RNAs (lncRNAs) have recently emerged as new regulatory molecules, with diverse functions in biological processes. In the present study, we found that expression of the ionizing radiation-inducible lncRNA, lnc-RI, was correlate negatively with micronucleus frequencies in human peripheral blood lymphocytes. Knockdown of lnc-RI significantly increased spontaneous DSBs levels, which was confirmed to be associated with the decreased efficiency of homologous recombination (HR) repair of DSBs. The expression of RAD51, a key recombinase in the HR pathway, decreased sharply in lnc-RI-depressed cells. In a further investigation, we demonstrated that miR-193a-3p could bind with both lnc-RI and RAD51 mRNA and depressed the expression of lnc-RI and RAD51 mRNA. Lnc-RI acted as a competitive endogenous RNA (ceRNA) to stabilize RAD51 mRNA via competitive binding with miR-193a-3p and release of its inhibition of RAD51 expression. To our knowledge, this is the first study to demonstrate the role of lnc-RI in regulating HR repair of DSBs. The feedback loop established in the current study suggests that lnc-RI is critical for the maintenance of genomic stability. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. Integrated miRNA, mRNA and protein expression analysis reveals the role of post-transcriptional regulation in controlling CHO cell growth rate

    Directory of Open Access Journals (Sweden)

    Clarke Colin

    2012-11-01

    Full Text Available Abstract Background To study the role of microRNA (miRNA in the regulation of Chinese hamster ovary (CHO cell growth, qPCR, microarray and quantitative LC-MS/MS analysis were utilised for simultaneous expression profiling of miRNA, mRNA and protein. The sample set under investigation consisted of clones with variable cellular growth rates derived from the same population. In addition to providing a systems level perspective on cell growth, the integration of multiple profiling datasets can facilitate the identification of non-seed miRNA targets, complement computational prediction tools and reduce false positive and false negative rates. Results 51 miRNAs were associated with increased growth rate (35 miRNAs upregulated and 16 miRNAs downregulated. Gene ontology (GO analysis of genes (n=432 and proteins (n=285 found to be differentially expressed (DE identified biological processes driving proliferation including mRNA processing and translation. To investigate the influence of miRNA on these processes we combined the proteomic and transcriptomic data into two groups. The first set contained candidates where evidence of translational repression was observed (n=158. The second group was a mixture of proteins and mRNAs where evidence of translational repression was less clear (n=515. The TargetScan algorithm was utilised to predict potential targets within these two groups for anti-correlated DE miRNAs. Conclusions The evidence presented in this study indicates that biological processes such as mRNA processing and protein synthesis are correlated with growth rate in CHO cells. Through the integration of expression data from multiple levels of the biological system a number of proteins central to these processes including several hnRNPs and components of the ribosome were found to be post-transcriptionally regulated. We utilised the expression data in conjunction with in-silico tools to identify potential miRNA-mediated regulation of m

  5. Integration of miRNA and mRNA expression profiles reveals microRNA-regulated networks during muscle wasting in cardiac cachexia

    DEFF Research Database (Denmark)

    Moraes, Leonardo N; Fernandez, Geysson J; Vechetti-Júnior, Ivan J

    2017-01-01

    Cardiac cachexia (CC) is a common complication of heart failure (HF) associated with muscle wasting and poor patient prognosis. Although different mechanisms have been proposed to explain muscle wasting during CC, its pathogenesis is still not understood. Here, we described an integrative analysis...... between miRNA and mRNA expression profiles of muscle wasting during CC. Global gene expression profiling identified 1,281 genes and 19 miRNAs differentially expressed in muscle wasting during CC. Several of these deregulated genes are known or putative targets of the altered miRNAs, including miR-29a-3p......, miR-29b-3p, miR-210-5p, miR-214, and miR-489. Gene ontology analysis on integrative mRNA/miRNA expression profiling data revealed miRNA interactions affecting genes that regulate extra-cellular matrix (ECM) organization, proteasome protein degradation, citric acid cycle and respiratory electron...

  6. Restricted distribution of mrg-1 mRNA in C. elegans primordial germ cells through germ granule-independent regulation.

    Science.gov (United States)

    Miwa, Takashi; Takasaki, Teruaki; Inoue, Kunio; Sakamoto, Hiroshi

    2015-11-01

    The chromodomain protein MRG-1 is an essential maternal factor for proper germline development that protects germ cells from cell death in C. elegans. Unlike germ granules, which are exclusively segregated to the germline blastomeres at each cell division from the first cleavage of the embryo, MRG-1 is abundant in all cells in early embryos and is then gradually restricted to the primordial germ cells (PGCs) by the morphogenesis stage. Here, we show that this characteristic spatiotemporal expression pattern is dictated by the mrg-1 3'UTR and is differentially regulated at the RNA level between germline and somatic cells. Asymmetric segregation of germ granules is not necessary to localize MRG-1 to the PGCs. We found that MES-4, an essential chromatin regulator in germ cells, also accumulates in the PGCs in a germ granule-independent manner. We propose that C.elegans PGCs have a novel mechanism to accumulate at least some chromatin-associated proteins that are essential for germline immortality. © 2015 The Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

  7. Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-transcriptional regulator, Hfq.

    Directory of Open Access Journals (Sweden)

    Alexandra Sittka

    2008-08-01

    Full Text Available Recent advances in high-throughput pyrosequencing (HTPS technology now allow a thorough analysis of RNA bound to cellular proteins, and, therefore, of post-transcriptional regulons. We used HTPS to discover the Salmonella RNAs that are targeted by the common bacterial Sm-like protein, Hfq. Initial transcriptomic analysis revealed that Hfq controls the expression of almost a fifth of all Salmonella genes, including several horizontally acquired pathogenicity islands (SPI-1, -2, -4, -5, two sigma factor regulons, and the flagellar gene cascade. Subsequent HTPS analysis of 350,000 cDNAs, derived from RNA co-immunoprecipitation (coIP with epitope-tagged Hfq or control coIP, identified 727 mRNAs that are Hfq-bound in vivo. The cDNA analysis discovered new, small noncoding RNAs (sRNAs and more than doubled the number of sRNAs known to be expressed in Salmonella to 64; about half of these are associated with Hfq. Our analysis explained aspects of the pleiotropic effects of Hfq loss-of-function. Specifically, we found that the mRNAs of hilD (master regulator of the SPI-1 invasion genes and flhDC (flagellar master regulator were bound by Hfq. We predicted that defective SPI-1 secretion and flagellar phenotypes of the hfq mutant would be rescued by overexpression of HilD and FlhDC, and we proved this to be correct. The combination of epitope-tagging and HTPS of immunoprecipitated RNA detected the expression of many intergenic chromosomal regions of Salmonella. Our approach overcomes the limited availability of high-density microarrays that have impeded expression-based sRNA discovery in microorganisms. We present a generic strategy that is ideal for the systems-level analysis of the post-transcriptional regulons of RNA-binding proteins and for sRNA discovery in a wide range of bacteria.

  8. Hungry granulocyte: its fate and regulation of production

    Energy Technology Data Exchange (ETDEWEB)

    Cronkite, E P

    1978-01-01

    The granulocyte, a phagocytic anti-1 bacterial defense cell, is discussed. Its production, the kinetics of its proliferation, the regulation of its production, and its loss from the blood are reviewed. (ACR)

  9. Federal approaches to the regulation of noncigarette tobacco products.

    Science.gov (United States)

    Freiberg, Michael J A

    2012-11-01

    Under a grant funded by ClearWay Minnesota(SM) and in partnership with nationally recognized experts in tobacco product regulation, the Public Health Law Center investigated how laws at every level apply, or fail to apply, to noncigarette tobacco products--also called "other tobacco products." During the years 2010-2011, standard legal research techniques were used to identify and compile relevant statutes, regulations, decisions, pleadings, proposals, and related materials. Sources included standard commercial legal databases such as LexisNexis and Westlaw, online sources for pending rules and legislation, and direct contact with courts for legal pleadings and unpublished decisions. These legal authorities related to many aspects of the regulation, including price, flavorants, youth access, marketing restrictions, and product design of other tobacco products. Five of these products were used as case studies: dissolvable tobacco products, electronic cigarettes, little cigars, snus, and water pipes. Research during the years 2010-2011 revealed that the federal regulation of other tobacco products lags behind the regulation of more "traditional" tobacco products, such as cigarettes and moist snuff. Federal regulatory options to expand regulation of these products were identified. The article highlights several federal policy interventions that would address gaps in the regulation of other tobacco products. The FDA must determine whether these interventions will benefit public health and, if so, to what extent--the legal criteria for intervention under the federal Family Smoking Prevention and Tobacco Control Act. Copyright © 2012 American Journal of Preventive Medicine. Published by Elsevier Inc. All rights reserved.

  10. Identification of TTP mRNA targets in human dendritic cells reveals TTP as a critical regulator of dendritic cell maturation

    Science.gov (United States)

    Emmons, Jillian; Townley-Tilson, W.H. Davin; Deleault, Kristen M.; Skinner, Stephen J.; Gross, Robert H.; Whitfield, Michael L.; Brooks, Seth A.

    2008-01-01

    Dendritic cells provide a critical link between innate and adaptive immunity and are essential to prime a naive T-cell response. The transition from immature dendritic cells to mature dendritic cells involves numerous changes in gene expression; however, the role of post-transcriptional changes in this process has been largely ignored. Tristetraprolin is an AU-rich element mRNA-binding protein that has been shown to regulate the stability of a number of cytokines and chemokines of mRNAs. Using TTP immunoprecipitations and Affymetrix GeneChips, we identified 393 messages as putative TTP mRNA targets in human dendritic cells. Gene ontology analysis revealed that ∼25% of the identified mRNAs are associated with protein synthesis. We also identified six MHC Class I alleles, five MHC Class II alleles, seven chemokine and chemokine receptor genes, indoleamine 2,3 dioxygenase, and CD86 as putative TTP ligands. Real-time PCR was used to validate the GeneChip data for 15 putative target genes and functional studies performed for six target genes. These data establish that TTP regulates the expression of DUSP1, IDO, SOD2, CD86, and MHC Class I-B and F via the 3′-untranslated region of each gene. A novel finding is the demonstration that TTP can interact with and regulate the expression of non-AU-rich element-containing messages. The data implicate TTP as having a broader role in regulating and limiting the immune response than previously suspected. PMID:18367721

  11. SCA8 mRNA expression suggests an antisense regulation of KLHL1 and correlates to SCA8 pathology.

    Science.gov (United States)

    Chen, Wei-Lun; Lin, Jun-Wei; Huang, Hei-Jen; Wang, Su-Min; Su, Ming-Tsan; Lee-Chen, Guey-Jen; Chen, Chiung-Mei; Hsieh-Li, Hsiu Mei

    2008-10-03

    An increasing number of inherited neurodegenerative diseases are known to be caused by the expansion of unstable trinucleotide repeat tracts. Spinocerebellar ataxia type 8 (SCA8) has been identified as being partly caused by a CTG expansion in an untranslated, endogenous antisense RNA that overlaps the Kelch-like 1 (KLHL1) gene. Clinically, SCA8 patients show similar features to those with the other SCAs, including limb and truncal ataxia, ataxic dysarthria and horizontal nystagmus, all of which are signs of dysfunction of the cerebellar system. However, allele sizes within the SCA8 proposed pathogenic range have been reported in patients with ataxia of unknown etiology, in individuals from pedigrees with other SCA or Friedreich's ataxia, and in patients with Alzheimer's disease, schizophrenia or parkinsonism. These observations suggest that mutation of the SCA8 locus might affect neurons other than the cerebellum. Antisense transcripts are known to regulate complementary sense transcripts and are involved in several biologic functions, such as development, adaptive response, and viral infection. In order to test whether SCA8 affects the KLHL1 expression by antisense RNA in brain cells, we examined the expression pattern of KLHL1 and SCA8 in human tissues and in mouse brain regions. SCA8 expression was colocalized with KLHL1 transcript in many brain regions whose functions are correlated to the clinical symptoms of SCA8 patients. These findings lead to the hypothesis of a possible relevance that SCA8 transcript downregulates KLHL1 expression through an antisense mechanism, which then leads to SCA8 neuropathogenesis.

  12. Central administration of antisense oligodeoxynucleotides to neuropeptide Y (NPY) mRNA reveals the critical role of newly synthesized NPY in regulation of LHRH release.

    Science.gov (United States)

    Kalra, P S; Bonavera, J J; Kalra, S P

    1995-10-20

    Compelling evidence shows that the episodic and cyclic secretion of hypothalamic luteinizing hormone releasing hormone (LHRH), the primary stimulator of pituitary LH release, is subject to regulation by neuropeptide Y (NPY). We have reported earlier that sequential treatment of ovariectomized (ovx) rats with estrogen and progesterone to stimulate a preovulatory-type LH surge elevated the levels of both NPY and preproNPY mRNA levels in the hypothalamus concomitant with dynamic changes in LHRH activity. The present study was designed to determine whether these elevations in NPY content and gene expression represent new synthesis of NPY that is crucial to elicit LHRH discharge. Ovx, steroid-primed rats received intracerebroventricular injections of an unmodified 20-mer oligodeoxynucleotide (oligo) complementary to the NPY mRNA sequence. Control rats were injected similarly with either saline or the sense or missense oligos. Results showed that control rats displayed a characteristic surge-type elevation in plasma LH levels that was not affected by the administration of missense or sense oligos. However, in rats injected with the antisense oligo, the steroid-induced LH surge was completely blocked. In an additional experiment, NPY peptide levels were measured in microdissected hypothalamic sites following the injection of antisense or missense oligos. NPY antisense oligo administration blocked the significant increases in NPY levels in the median eminence-arcuate area, the medial preoptic area and lateral preoptic area seen in control rats. These results suggest that sequential ovarian steroid treatment augments NPY synthesis in the hypothalamus and this newly synthesized NPY is critical for induction of the LHRH and LH surge.

  13. Differential mRNA expression and glucocorticoid-mediated regulation of TRPM6 and TRPM7 in the heart and kidney throughout murine pregnancy and development.

    Directory of Open Access Journals (Sweden)

    James S M Cuffe

    Full Text Available The transient receptor potential (TRP channels TRPM6 and TRPM7 are critically involved in maintaining whole body and cellular Mg2+ homeostasis and ensuring the normal function of organs such as the heart and kidney. However, we do not know how the expression of TRPM6 and TPRM7 in these organs changes throughout fetal development and adult life, and whether this expression can be hormonally regulated. This study determined the ontogeny of TRPM6 and TRPM7 mRNA expression from mid-gestation through to adulthood in the mouse. In a second series of experiments, we examined how maternal administration of the glucocorticoids corticosterone and dexamethasone between embryonic days 12.5-15 affected TRPM6 and TRPM7 channel mRNA expression in the mother and fetus. Whilst renal TRPM7 expression was relatively constant throughout development, renal TRPM6 expression was markedly upregulated after birth. In contrast, cardiac TRPM7 expression was 2-4 fold higher in the fetus than in the adult. Surprisingly, TRPM6 expression was detected in the fetal heart (qPCR and in situ hybridization. Glucocorticoid administration during gestation increased fetal cardiac expression of both channels without affecting renal expression. In contrast, in the dam renal TRPM6 and TRPM7 expression was increased by glucocorticoids with no change in the cardiac channel expression. These data suggest that TRPM6 and TRPM7 channels are important in organogenesis, and that elevated maternal glucocorticoid levels can alter the expression of these channels. This suggests that perturbations in hormonal regulatory systems during pregnancy may adversely impact upon normal fetal development, at least in part by altering expression of TRPM channels.

  14. Silibinin down-regulates survivin protein and mRNA expression and causes caspases activation and apoptosis in human bladder transitional-cell papilloma RT4 cells.

    Science.gov (United States)

    Tyagi, Anil K; Agarwal, Chapla; Singh, Rana P; Shroyer, Kenneth R; Glode, L Michael; Agarwal, Rajesh

    2003-12-26

    Bladder cancer is the fourth and eighth most common cancer in men and women in the United States, respectively. Survivin, a member of inhibitor of apoptosis protein (IAP) gene family, is deregulated in a wide range of malignancies, including carcinoma of the bladder urothelium. Recent advances have identified survivin as a novel intervention target to induce apoptosis in cancer cells by phytochemicals or synthetic agents. Silibinin is a naturally occurring flavanone, isolated from milk thistle extract, and has been shown to possess cancer prevention/intervention potential against various cancers. In several animal and human studies, it is found to be safe and non-toxic. Human bladder transitional-cell papilloma RT4 cells were treated with silibinin and analyzed for survivin protein and mRNA levels by Western blotting and real-time RT-PCR, respectively. Silibinin treatment of cells for 24 h at 100 microM dose resulted in approximately 50% decrease in survivin protein level; however, treatment at 200 microM dose for 24 and 48 h showed a complete loss in survivin protein without any change in actin used as loading control. Employing RT-PCR analysis we also observed that silibinin causes a strong to complete decrease in survivin mRNA levels. In other studies, down-regulation of survivin by silibinin was associated with a very strong and prominent caspases-9 and -3 activation as well as PARP cleavage. Quantitative apoptotic assay showed that silibinin decreased survivin levels and caspases-PARP cleavages, in accord with a strong apoptotic death and growth inhibition of RT4 cells. Together, these findings suggest that more studies are needed to investigate in vivo effect of silibinin on survivin expression and associated biological effects in bladder cancer that could provide useful information for silibinin efficacy in the prevention/intervention of human bladder cancer.

  15. Developmental expression of the protein product of Vg1, a localized maternal mRNA in the frog Xenopus laevis.

    OpenAIRE

    Dale, L; Matthews, G; Tabe, L; Colman, A

    1989-01-01

    Vg1 is a maternal mRNA localized in the vegetal cortex of Xenopus laevis oocytes, that encodes a protein homologous to the mammalian growth factor TGF-beta. Using a polyclonal antibody to a T7-Vg1 fusion protein, we have identified the native protein. We find that a single protein of Mr 40 kd is immunoprecipitated following in vitro translation of oocyte poly(A)+ RNA, whilst two proteins of Mr 45 and 43.5 kd are immunoprecipitated from oocyte and embryo extracts. Synthesis of at least the 40 ...

  16. Nitrous oxide production and mRNA expression analysis of nitrifying and denitrifying bacterial genes under floodwater disappearance and fertilizer application.

    Science.gov (United States)

    Riya, Shohei; Takeuchi, Yuki; Zhou, Sheng; Terada, Akihiko; Hosomi, Masaaki

    2017-06-01

    A pulse of nitrous oxide (N2O) emission has been observed following the disappearance of floodwater by drainage. However, its mechanism is not well understood. We conducted a column study to clarify the mechanism for N2O production during floodwater disappearance by using a microsensor and determining the bacterial gene expression. An increase in N2O flux was observed following floodwater disappearance after the addition of NH4+, with a corresponding increase in the concentrations of NO3- and dissolved N2O in the oxic and anoxic soil layers, respectively. The transcription level of the bacterial amoA mRNA did not change, while that of nirK mRNA increased sharply after an hour of floodwater disappearance. An additional anoxic soil slurry experiment demonstrated that the addition of NO3- induced the expression of nirK gene and caused a concomitant increase in N2O production. These findings suggest that NO3- production in the oxic layers is important as it provides a substrate and induces the synthesis of denitrification enzymes in the anoxic layer during N2O production.

  17. Regulation of LacZ mRNA translatability in a cell-free system at heat shock by the last four sense codons.

    Science.gov (United States)

    Denisenko, O N; Yarchuk, O B

    1989-04-24

    Under heat shock conditions translation of Xenopus laevis normal mRNAs in a rabbit reticulocyte cell-free system is blocked whereas hsp70 mRNA is translated. mRNA for E. coli beta-galactosidase containing the last four sense codons of Drosophila hsp70 at its 3'-end was constructed. This mRNA is efficiently translated in a rabbit reticulocyte cell-free system at 43 degrees C.

  18. Foetal life protein provision of mink (Neovison vison) changes the relative mRNA abundance of some hepatic enzymes regulating fat metabolism.

    Science.gov (United States)

    Matthiesen, Connie Frank; Casañas, Maria Arantzazu Aguinaga; Tauson, Anne-Helene

    2014-01-01

    The nutrient provision to pregnant females has high impact on the growth and metabolism of their offspring. The objective was to investigate if the expression of hepatic enzymes regulating the fat metabolism was affected in foetuses and adult female mink born by dams fed either a low or an adequate level of protein during late gestation. The relative abundances of acetyl coenzyme A carboxylase (ACC), fatty acid synthase (FAS) and carnitine palmitoyl transferase 1 (CPT1) mRNA were determined by qualitative polymerase chain reaction in the livers of F₀- and F₁-generation dams and in F₁-generation foetuses. Low protein provision during foetal life resulted in a lower expression of FAS in foetal liver but a tendency towards increased expression in the liver of adult dams. There was a tendency towards an effect of life stage of the animal on the expression of ACC resulting in a higher expression among F₁ foetuses exposed to low protein during foetal life than F₀ dams fed a low protein diet during late gestation. The expression of CPT1 was significantly lower among dams exposed to low protein provision during foetal life than controls, possibly indicating a lower rate of mitochondrial β-oxidation. Further investigations are needed to clarify the consequences of these changes for the fat metabolism.

  19. The staphylococcal accessory regulator, SarA, is an RNA-binding protein that modulates the mRNA turnover properties of late-exponential and stationary phase Staphylococcus aureus cells

    Directory of Open Access Journals (Sweden)

    John M Morrison

    2012-03-01

    Full Text Available The modulation of mRNA turnover is gaining recognition as a mechanism by which Staphylococcus aureus regulates gene expression, but the factors that orchestrate alterations in transcript degradation are poorly understood. In that regard, we previously found that 138 mRNA species, including the virulence factors protein A (spa and collagen binding protein (cna, are stabilized in a sarA-dependent manner during exponential phase growth, suggesting that SarA protein may directly or indirectly effect the RNA turnover properties of these transcripts. Herein, we expanded our characterization of the effects of sarA on mRNA turnover during late exponential and stationary phases of growth. Results revealed that the locus affects the RNA degradation properties of cells during both growth phases. Further, using gel mobility shift assays and RIP-ChIP, it was found that SarA protein is capable of binding mRNA species that it stabilizes both in vitro and within bacterial cells. Taken together, these results suggest that SarA post-transcriptionally regulates S. aureus gene expression in a manner that involves binding to and consequently altering the mRNA turnover properties of target transcripts.

  20. Up-Regulation of mRNA Ventricular PRNP Prion Protein Gene Expression in Air Pollution Highly Exposed Young Urbanites: Endoplasmic Reticulum Stress, Glucose Regulated Protein 78, and Nanosized Particles

    Directory of Open Access Journals (Sweden)

    Rodolfo Villarreal-Calderon

    2013-11-01

    Full Text Available Mexico City Metropolitan Area children and young adults exposed to high concentrations of air pollutants including fine and ultrafine particulate matter (PM vs. clean air controls, exhibit myocardial inflammation and inflammasome activation with a differential right and left ventricular expression of key inflammatory genes and inflammasomes. We investigated the mRNA expression levels of the prion protein gene PRNP, which plays an important role in the protection against oxidative stress and metal toxicity, and the glucose regulated protein 78, a key protein in endoplasmic reticulum (ER stress signaling, in ventricular autopsy samples from 30 children and young adults age 19.97 ± 6.8 years with a lifetime of low (n:4 vs. high (n:26 air pollution exposures. Light microscopy and transmission electron microscopy studies were carried out in human ventricles, and electron microscopy studies were also done in 5 young, highly exposed Mexico City dogs. There was significant left ventricular PRNP and bi-ventricular GRP78 mRNA up-regulation in Mexico City young urbanites vs. controls. PRNP up-regulation in the left ventricle was significantly different from the right, p < 0.0001, and there was a strong left ventricular PRNP and GRP78 correlation (p = 0.0005. Marked abnormalities in capillary endothelial cells, numerous nanosized particles in myocardial ER and in abnormal mitochondria characterized the highly exposed ventricles. Early and sustained cardiac ER stress could result in detrimental irreversible consequences in urban children, and while highly complex systems maintain myocardial homeostasis, failure to compensate for chronic myocardial inflammation, oxidative and ER stress, and particles damaging myocardial organelles may prime the development of pathophysiological cardiovascular states in young urbanites. Nanosized PM could play a key cardiac myocyte toxicity role.

  1. Some modern notions on oil and gas reservoir production regulation

    Energy Technology Data Exchange (ETDEWEB)

    Lohrenz, J.; Monash, E.A.

    1980-05-21

    The historic rhetoric of oil and gas reservoir production regulations has been burdened with misconceptions. One was that most reservoirs are rate insensitive. Another was that a reservoir's decline is primarily a function of reservoir mechaism rather than a choice unconstrained by the laws of physics. Relieved of old notions like these, we introduce some modern notions, the most basic being that production regulation should have the purpose of obtaining the highest value from production per irreversible diminution of thermodynamically available energy. The laws of thermodynamics determine the available energy. What then is value. Value may include contributions other than production per se and purely monetary economic outcomes.

  2. Toward National Uniformity for FDA-Regulated Products

    OpenAIRE

    Semet, Amy E.

    2000-01-01

    In this paper, I will argue that national uniformity should be granted to all FDA-regulated products and should include preemption of both state labeling and safety standards as well as state product liability laws. Cosmetics and food should be treated the same as FDAMA treats OTC drugs. Although the FDA has developed different regulatory regimes for each product, the FDA has established the optimal standards for all of its products, and states should not interfere to preclude harmonization. ...

  3. The Me31B DEAD-box helicase localizes to postsynaptic foci and regulates expression of a CaMKII reporter mRNA in dendrites of Drosophila olfactory projection neurons

    Directory of Open Access Journals (Sweden)

    Jens Hillebrand

    2010-11-01

    Full Text Available mRNP granules at adult central synapses are postulated to regulate local mRNA translation and synapse plasticity. However, they are very poorly characterized in vivo. Here, in Drosophila olfactory synapses, we present early observations and characterization of candidate synaptic mRNP particles, one of which contains a widely conserved, DEAD-box helicase, Me31B. In Drosophila, Me31B is required for translational repression of maternal and miRNA-target mRNAs. A role in neuronal translational control is primarily suggested by Me31B’s localization, in cultured primary neurons, to neuritic mRNP granules that contain: i various translational regulators; ii CaMKII mRNA; and iii several P-body markers including the mRNA hydrolases, Dcp1 and Pcm/Xrn-1. In adult neurons, Me31B localizes to P-body like cytoplasmic foci/particles in neuronal soma. In addition it is present to synaptic foci that may lack RNA degradative enzymes and localize predominantly to dendritic elements of olfactory sensory and projection neurons. MARCM clones of projection-neurons mutant for Me31B show loss of both Me31B and Dcp1-positive dendritic puncta, suggesting potential interactions between these granule types. In projection neurons, expression of validated hairpin-RNAi constructs against Me31B causes visible knockdown of endogenous protein, as assessed by the brightness and number of Me31B puncta. Knockdown of Me31B also causes a substantial elevation in observed levels of a translational reporter of CaMKII, a postsynaptic protein whose mRNA has been shown to be localized to projection neuron dendrites and to be translationally regulated, at least in part through the miRNA pathway. Thus, neuronal Me31B is present in dendritic particles in vivo and is required for repression of a translationally regulated synaptic mRNA.

  4. Epstein-Barr virus mRNA export factor EB2 is essential for intranuclear capsid assembly and production of gp350.

    Science.gov (United States)

    Batisse, Julien; Manet, Evelyne; Middeldorp, Jaap; Sergeant, Alain; Gruffat, Henri

    2005-11-01

    Most human herpesviruses, including Epstein-Barr virus (EBV), express a protein which functions primarily as an mRNA export factor. Previously, we deleted the gene for the Epstein-Barr virus mRNA export factor EB2 from the EBV genome and then introduced the mutated genome into 293 cells. Using a transcomplementation assay in which ectopic expression of the transcription factor EB1/ZEBRA was sufficient to induce the EBV productive cycle, we showed that Ori-Lyt-dependent replication of the EBV DNA occurs in the absence of EB2, indicating that EB2 is not essential for the expression and export of early mRNAs. However, in the absence of EB2, no infectious viral particles are produced (H. Gruffat, J. Batisse, D. Pich, B. Neuhierl, E. Manet, W. Hammerschmidt, and A. Sergeant, J. Virol. 76:9635-9644, 2002). In this report, we now show that EB2 is essential for the nuclear export of most, but not all, late mRNAs produced from intronless genes that translate into proteins involved in intranuclear capsid assembly and maturation. As a consequence, we show that EB2 is essential for the proper assembly of intranuclear capsids. Interestingly, the late BLLF1 gene contains an intron, and both unspliced and spliced mRNAs must be exported to the cytoplasm to be translated into gp350 and gp220, respectively (M. Hummel, D. A. Thorley-Lawson, and E. Kieff, J. Virol. 49:413-417, 1984). Our results also demonstrate that although BLLF1 spliced mRNAs are exported to the cytoplasm independently of EB2, EB2 is essential for the nuclear export of unspliced BLLF1 mRNA. In the same assay, herpes simplex virus 1 ICP27 completely inhibited the nuclear export of BLLF1 spliced mRNAs whereas unspliced BLLF1 mRNAs were exported, confirming that in a physiological assay, ICP27 inhibits splicing.

  5. RNA-binding protein regulates plant DNA methylation by controlling mRNA processing at the intronic heterochromatin-containing gene IBM1.

    Science.gov (United States)

    Wang, Xingang; Duan, Cheng-Guo; Tang, Kai; Wang, Bangshing; Zhang, Huiming; Lei, Mingguang; Lu, Kun; Mangrauthia, Satendra K; Wang, Pengcheng; Zhu, Guohui; Zhao, Yang; Zhu, Jian-Kang

    2013-09-17

    DNA methylation-dependent heterochromatin formation is a conserved mechanism of epigenetic silencing of transposons and other repeat elements in many higher eukaryotes. Genes adjacent to repetitive elements are often also subjected to this epigenetic silencing. Consequently, plants have evolved antisilencing mechanisms such as active DNA demethylation mediated by the REPRESSOR OF SILENCING 1 (ROS1) family of 5-methylcytosine DNA glycosylases to protect these genes from silencing. Some transposons and other repeat elements have found residence in the introns of genes. It is unclear how these intronic repeat elements-containing genes are regulated. We report here the identification of ANTI-SILENCING 1 (ASI1), a bromo-adjacent homology domain and RNA recognition motif-containing protein, from a forward genetic screen for cellular antisilencing factors in Arabidopsis thaliana. ASI1 is required to prevent promoter DNA hypermethylation and transcriptional silencing of some transgenes. Genome-wide DNA methylation analysis reveals that ASI1 has a similar role to that of the histone H3K9 demethylase INCREASE IN BONSAI METHYLATION 1 (IBM1) in preventing CHG methylation in the bodies of thousands of genes. We found that ASI1 is an RNA-binding protein and ensures the proper expression of IBM1 full-length transcript by associating with an intronic heterochromatic repeat element of IBM1. Through mRNA sequencing, we identified many genes containing intronic transposon elements that require ASI1 for proper expression. Our results suggest that ASI1 associates with intronic heterochromatin and binds the gene transcripts to promote their 3' distal polyadenylation. The study thus reveals a unique mechanism by which higher eukaryotes deal with the collateral effect of silencing intronic repeat elements.

  6. Manipulation of the carbon storage regulator system for metabolite remodeling and biofuel production in Escherichia coli

    Science.gov (United States)

    2012-01-01

    Background Microbial engineering strategies that elicit global metabolic perturbations have the capacity to increase organism robustness for targeted metabolite production. In particular, perturbations to regulators of cellular systems that impact glycolysis and amino acid production while simultaneously decreasing fermentation by-products such as acetate and CO2 make ideal targets. Intriguingly, perturbation of the Carbon Storage Regulator (Csr) system has been previously implicated in large changes in central carbon metabolism in E. coli. Therefore, we hypothesized that perturbation of the Csr system through the CsrA-CsrB ribonucleoprotein complex might increase production of biofuels and their intermediates from heterologous pathways. Results We engaged the CsrA-CsrB ribonucleoprotein complex of E. coli via overexpression of CsrB. CsrB is a 350-nucleotide non-coding RNA that antagonizes CsrA, an RNA-binding protein that regulates translation of specific mRNA targets. By using shotgun proteomics and targeted metabolomics we established that elevation of CsrB levels leads to alterations in metabolite and protein levels in glycolysis, the TCA cycle and amino acid levels. Consequently, we show that such changes can be suitably applied to improve the production of desired compounds through the native fatty acid and heterologous n-butanol and isoprenoid pathways by up to two-fold. We also observed concomitant decreases in undesirable fermentation by-products such as acetate and CO2. Conclusions We have demonstrated that simple engineering of the RNA-based Csr global regulatory system constitutes a novel approach to obtaining pathway-independent improvements within engineered hosts. Additionally, since Csr is conserved across most prokaryotic species, this approach may also be amenable to a wide variety of production hosts. PMID:22694848

  7. SMG-8 and SMG-9, two novel subunits of the SMG-1 complex, regulate remodeling of the mRNA surveillance complex during nonsense-mediated mRNA decay

    Science.gov (United States)

    Yamashita, Akio; Izumi, Natsuko; Kashima, Isao; Ohnishi, Tetsuo; Saari, Bonnie; Katsuhata, Yukiko; Muramatsu, Reiko; Morita, Tomoko; Iwamatsu, Akihiro; Hachiya, Takahisa; Kurata, Rie; Hirano, Hisashi; Anderson, Philip; Ohno, Shigeo

    2009-01-01

    Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that detects and degrades mRNAs containing premature translation termination codons (PTCs). SMG-1 and Upf1 transiently form a surveillance complex termed “SURF” that includes eRF1 and eRF3 on post-spliced mRNAs during recognition of PTC. If an exon junction complex (EJC) exists downstream from the SURF complex, SMG-1 phosphorylates Upf1, the step that is a rate-limiting for NMD. We provide evidence of an association between the SURF complex and the ribosome in association with mRNPs, and we suggest that the SURF complex functions as a translation termination complex during NMD. We identified SMG-8 and SMG-9 as novel subunits of the SMG-1 complex. SMG-8 and SMG-9 suppress SMG-1 kinase activity in the isolated SMG-1 complex and are involved in NMD in both mammals and nematodes. SMG-8 recruits SMG-1 to the mRNA surveillance complex, and inactivation of SMG-8 induces accumulation of a ribosome:Upf1:eRF1:eRF3:EJC complex on mRNP, which physically bridges the ribosome and EJC through eRF1, eRF3, and Upf1. These results not only reveal the regulatory mechanism of SMG-1 kinase but also reveal the sequential remodeling of the ribosome:SURF complex to the predicted DECID (DECay InDucing) complex, a ribosome:SURF:EJC complex, as a mechanism of in vivo PTC discrimination. PMID:19417104

  8. TRIM11 negatively regulates IFNβ production and antiviral activity by targeting TBK1.

    Directory of Open Access Journals (Sweden)

    Younglang Lee

    Full Text Available The innate immune response is a host defense mechanism against infection by viruses and bacteria. Type I interferons (IFNα/β play a crucial role in innate immunity. If not tightly regulated under normal conditions and during immune responses, IFN production can become aberrant, leading to inflammatory and autoimmune diseases. In this study, we identified TRIM11 (tripartite motif containing 11 as a novel negative regulator of IFNβ production. Ectopic expression of TRIM11 decreased IFNβ promoter activity induced by poly (I:C stimulation or overexpression of RIG-I (retinoic acid-inducible gene-I signaling cascade components RIG-IN (constitutively active form of RIG-I, MAVS (mitochondrial antiviral signaling protein, or TBK1 (TANK-binding kinase-1. Conversely, TRIM11 knockdown enhanced IFNβ promoter activity induced by these stimuli. Moreover, TRIM11 overexpression inhibited the phosphorylation and dimerization of IRF3 and expression of IFNβ mRNA. By contrast, TRIM11 knockdown increased the IRF3 phosphorylation and IFNβ mRNA expression. We also found that TRIM11 and TBK1, a key kinase that phosphorylates IRF3 in the RIG-I pathway, interacted with each other through CC and CC2 domain, respectively. This interaction was enhanced in the presence of the TBK1 adaptor proteins, NAP1 (NF-κB activating kinase-associated protein-1, SINTBAD (similar to NAP1 TBK1 adaptor or TANK (TRAF family member-associated NF-κB activator. Consistent with its inhibitory role in RIG-I-mediated IFNβ signaling, TRIM11 overexpression enhanced viral infectivity, whereas TRIM11 knockdown produced the opposite effect. Collectively, our results suggest that TRIM11 inhibits RIG-I-mediated IFNβ production by targeting the TBK1 signaling complex.

  9. Diazepam enhances production of diazepam-binding inhibitor (DBI), a negative saliva secretion regulator, localized in rat salivary gland.

    Science.gov (United States)

    Tsukagoshi, Eri; Kawaguchi, Mitsuru; Shinomiya, Takashi; Yoshikawa, Masanobu; Kawano, Toshihiko; Okubo, Migiwa; Sawaki, Kohei

    2011-01-01

    Peripheral-type benzodiazepine receptor (PBR) and central-type benzodiazepine receptor (CBR) in salivary gland play a role in the inhibitory regulation of salivary secretion in rodents. Diazepam-binding inhibitor (DBI), an endogenous ligand for PBR, produces neurosteroids, which modulate CBR activity. In this study, we investigated the effect of repetitive administration of diazepam (DZP) on salivary secretion and expression of DBI mRNA and peptide. Moreover, mRNA expression of PBR and pituitary adenylate cyclase-activating polypeptide (PACAP), a transcriptional regulator for DBI promoter, was evaluated after repetitive administration of DZP. Repetitive administration, but not single administration, of 0.4 mg/kg DZP caused inhibition of salivary secretion and enhanced expression of DBI, PACAP, and PBR mRNA in rat salivary gland, with an increase in production of DBI peptide. These results suggest that repetitive administration of DZP stimulates DBI production, which may result in an increase in the suppressive effect of DZP on salivary secretion.

  10. Federal Environmental Regulations Impacting Hydrocarbon Exploration, Drilling, and Production Operations

    Energy Technology Data Exchange (ETDEWEB)

    Carroll, Herbert B.; Johnson, William I.

    1999-04-27

    Waste handling and disposal from hydrocarbon exploration, drilling, and production are regulated by the US Environmental Protection Agency (EPA) through federal and state regulations and/or through implementation of federal regulations. Some wastes generated in these operations are exempt under the Resource Conservation and Recovery Act (RCRA) but are not exempt under the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA), Superfund Amendments and Reauthorization Act (SARA), and other federal environmental laws. Exempt wastes remain exempt only if they are not mixed with hazardous wastes or hazardous substances. Once mixture occurs, the waste must be disposed as a hazardous material in an approved hazardous waste disposal facility. Before the Clean Air Act as amended in 1990, air emissions from production, storage, steam generation, and compression facilities associated with hydrocarbon exploration, drilling, and production industry were not regulated. A critical proposed regulatory change which will significantly effect Class II injection wells for disposal of produced brine and injection for enhanced oil recovery is imminent. Federal regulations affecting hydrocarbon exploration, drilling and production, proposed EPA regulatory changes, and a recent significant US Court of Appeals decision are covered in this report. It appears that this industry will, in the future, fall under more stringent environmental regulations leading to increased costs for operators.

  11. Effects of antihistamine on up-regulation of histamine H1 receptor mRNA in the nasal mucosa of patients with pollinosis induced by controlled cedar pollen challenge in an environmental exposure unit

    Directory of Open Access Journals (Sweden)

    Yoshiaki Kitamura

    2015-11-01

    Full Text Available In the present study, we examined the effects of antihistamine on the up-regulation of H1R mRNA in the nasal mucosa of patients with pollinosis induced by controlled exposure to pollen using an environmental exposure unit. Out of 20 patients, we designated 14 responders, whose levels of H1R mRNA in the nasal mucosa were increased after the first pollen exposure and excluded 6 non-responders. Accordingly, the first exposure to pollen without treatment significantly induced both nasal symptoms and the up-regulation of H1R mRNA in the nasal mucosa of the responders. Subsequently, prophylactic administration of antihistamine prior to the second pollen exposure significantly inhibited both of the above effects in the responders. Moreover, the nasal expression of H1R mRNA before the second pollen exposure in the responders pretreated with antihistamine was significantly decreased, as compared with that before the first pollen exposure without treatment. These findings suggest that antihistamines suppressed histamine-induced transcriptional activation of H1R gene in the nasal mucosa, in addition to their blocking effect against histamine on H1R, resulting in a decrease of nasal symptoms. These findings further suggest that by their inverse agonistic activity, antihistamines suppress the basal transcription of nasal H1R in the absence of histamine in responders.

  12. BlpC-regulated bacteriocin production in Streptococcus thermophilus.

    Science.gov (United States)

    Renye, John A; Somkuti, George A

    2013-03-01

    Streptococcus thermophilus B59671 produces a bacteriocin with anti-pediococcal activity, but genes required for its production are not characterized. Genome sequencing of S. thermophilus has identified a genetic locus encoding a quorum sensing (QS) system that regulates production of class II bacteriocins. However, in strains possessing this gene cluster, production of bacteriocin like peptides (Blp) was only observed when excess pheromone was provided. PCR analysis revealed this strain possessed blpC, which encodes the 30-mer QS pheromone. To investigate if BlpC regulates bacteriocin production in S. thermophilus B59671, an integrative vector was used to replace blpC with a gene encoding for kanamycin resistance and the resulting mutant did not inhibit the growth of Pediococcus acidilactici. Constitutive expression of blpC from a shuttle vector restored the bacteriocin production, confirming the blp gene cluster is essential for bacteriocin activity in S. thermophilus B59671.

  13. Regulation of dual oxidase expression and H2O2 production by thyroglobulin.

    Science.gov (United States)

    Yoshihara, Aya; Hara, Takeshi; Kawashima, Akira; Akama, Takeshi; Tanigawa, Kazunari; Wu, Huhehasi; Sue, Mariko; Ishido, Yuko; Hiroi, Naoki; Ishii, Norihisa; Yoshino, Gen; Suzuki, Koichi

    2012-10-01

    Thyroglobulin (Tg) is a macromolecular precursor in thyroid hormone synthesis to which iodine is stably bound. Tg, which is stored in the follicular space, is also a potent negative feedback regulator of follicular function, and this is achieved by suppressing mRNA levels of thyroid-specific genes such as the sodium/iodide symporter (Slc5a5), Tg, and thyroid peroxidase. Dual oxidase 1 (DUOX1) and DUOX2, originally identified in the thyroid, are nicotinamide adenine dinucleotide phosphate (NADPH) oxidases that are necessary to produce the H2O2 required for thyroid hormone biosynthesis. Since follicular Tg regulates the expression of genes that are essential for thyroid hormone synthesis, we hypothesized that Tg might also regulate DUOX expression and H2O2 production. Rat thyroid FRTL-5 cells were treated with Tg, and the mRNA expression of Duox1 and Duox2 and their corresponding maturation factors Duoxa1 and Duoxa2 were evaluated by DNA microarray and real-time PCR. Duox2 promoter activity was examined by luciferase reporter gene assay. Protein levels of DUOX2 were also examined by Western blot analysis. Intracellular H2O2 generation was quantified by a fluorescent dye, 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, and acetyl ester (CM-H2DCFDA). mRNA levels of Duox2 and its activation factor Duoxa2 (but not Duox1 or Duoxa1) were significantly suppressed by Tg in a dose-dependent manner and a time-dependent fashion in rat thyroid FRTL-5 cells. DUOX2 promoter activity was significantly suppressed by Tg in a dose-dependent manner. Protein levels of DUOX2 and H2O2 generation in cells were also reduced by Tg treatment. We show that physiological concentrations of Tg suppressed the expression and function of DUOX2 in thyroid cells. These results suggest that Tg is a strong suppressor of the expression and the activity of DUOX2/DUOXA2, thereby regulating iodide organification and hormone synthesis in the thyroid. The evidence supports a reported model

  14. Regulation of secondary metabolite production in filamentous ascomycetes.

    Science.gov (United States)

    Shwab, E Keats; Keller, Nancy P

    2008-02-01

    Fungi are renowned for their ability to produce bioactive small molecules otherwise known as secondary metabolites. These molecules have attracted much attention due to both detrimental (e.g. toxins) and beneficial (e.g. pharmaceuticals) effects on human endeavors. Once the topic only of chemical and biochemical studies, secondary metabolism research has reached a sophisticated level in the realm of genetic regulation. This review covers the latest insights into the processes regulating secondary metabolite production in filamentous fungi.

  15. The Canadian Natural Health Products (NHP) Regulations: Industry Compliance Motivations

    OpenAIRE

    Hina Laeeque; Heather Boon; Natasha Kachan; Jillian Clare Cohen; Joseph D'Cruz

    2006-01-01

    This qualitative study explores corporations' motivations to comply with new natural health products (NHP) Regulations in Canada. Interviews were conducted with representatives from 20 Canadian NHP companies. Findings show that the rationale for compliance differs for large compared to small and medium-sized enterprises (SMEs). Large firms are motivated to comply with the regulations because of the deterrent fear of negative media coverage, social motivations, ability to comply and maintainin...

  16. Seasonal regulation of primary production in eastern boundary upwelling systems

    Science.gov (United States)

    Messié, Monique; Chavez, Francisco P.

    2015-05-01

    The regulation of seasonal satellite-derived primary production (PP) was investigated within a 150 km coastal box in four eastern boundary upwelling systems (EBUS): California, Peru, Northwest Africa and Benguela. The following regulating factors were considered: (1) wind-driven nitrate supply; (2) iron supply inferred from proxies (shelf mud belt width, modeled atmospheric iron deposition, river discharge); (3) temperature; (4) light and (5) physical export consisting of offshore export, eddy-driven and wind-driven subduction. The ratio of potential new production (carbon-equivalent of nitrate supply) to primary production, termed the N-ratio, is shown to be an indicator of PP limitation by nitrate supply (low N-ratios) vs. inhibition by other factors (high N-ratios). The factors regulating PP were assessed by analyzing the N-ratios and computing spatial correlations between PP and each factor each month. The regulation of primary production was found to vary spatially, seasonally and from one EBUS to another. Macronutrient supply is shown to be the dominant regulating factor off Northwest Africa and during some seasons and locations in other systems. Light regulation within the mixed layer occurs in all EBUS in winter but may only inhibit PP (high N-ratios) off Peru and Benguela. Evidence for iron limitation was found in each EBUS (except Northwest Africa) at varying levels and was greatest off Peru during austral winter when iron demand by phytoplankton increases due to low light levels. Rapid offshore advection combined with wind-driven and/or eddy-driven subduction may inhibit PP off California. A simple generalization regarding the regulation of primary production in EBUS is not forthcoming.

  17. Molecular cloning of type I collagen cDNA and nutritional regulation of type I collagen mRNA expression in grass carp.

    Science.gov (United States)

    Yu, E M; Liu, B H; Wang, G J; Yu, D G; Xie, J; Xia, Y; Gong, W B; Wang, H H; Li, Z F; Wei, N

    2014-08-01

    Grass carp (Ctenopharyngodon idellus) are important Chinese freshwater fish, and in China, the faba bean has been used as the sole food source for grass carp to transform them into crisp grass carp. Because of this, crisp grass carp has become an economically important fish because of its increased muscle hardness. To study the nutritional regulation of type I collagen in faba bean-fed grass carp, we isolated type I collagen alpha 2 (COL1A2) on the basis of our isolation of COL1A1. The COL1A2 cDNA was found to be 4899 bp in length and included a 4059-bp coding sequence (CDS) and encoded a polypeptide of 1352 AA. The protein peptide molecular weight was 127.39 kD, and the theoretical isoelectric point was 9.37. The COL1A2 protein possessed five α-helixes, eight β-sheets, 16 regions of triple helical repeats, 21 low-complexity regions, 10 function domains and two zinc-binding sites; however, no calcium-binding sites were observed. The mRNA expression of COL1A1 and COL1A2 was assessed in eight tissues (muscle, hepatopancreas, intestine, gills, skin, fin, kidney and spleen) from grass carp and crisp grass carp by semi-quantitative RT-PCR. Expression of COL1A1 in the muscle, intestines and skin of crisp grass carp was higher than that in grass carp, and expression of COL1A2 in the muscle, gills, fin and skin of crisp grass carp was higher than that in grass carp. In the muscle of crisp grass carp, expression of COL1A1 and COL1A2 was higher than that in grass carp, which was further confirmed by real-time PCR, and collagen content also was enhanced. These results demonstrated that type I collagen was closely related to the increased muscle hardness of faba bean-fed grass carp. Journal of Animal Physiology and Animal Nutrition © 2013 Blackwell Verlag GmbH.

  18. Regulation of lysine catabolism in Arabidopsis through concertedly regulated synthesis of the two distinct gene products of the composite AtLKR/SDH locus.

    Science.gov (United States)

    Stepansky, Asya; Yao, Youli; Tang, Guiliang; Galili, G

    2005-02-01

    Lysine catabolism in plants is initiated by a bifunctional LKR/SDH (lysine-ketoglutarate reductase/saccharopine dehydrogenase) enzyme encoded by a single LKR/SDH gene. Yet, the AtLKR/SDH gene of Arabidopsis also encodes a second gene product, namely a monofunctional SDH. To elucidate the regulation of lysine catabolism in Arabidopsis through these two gene products of the AtLKR/SDH gene, an analysis was carried out on the effects of the hormones, abscisic acid and jasmonate, as well as various metabolic and stress signals, including lysine itself, on their mRNA and protein levels. The response of the two gene products to the various treatments was only partially co-ordinated, but the levels of the monofunctional SDH mRNA and protein were always in excess over their bifunctional LKR/SDH counterparts. These results suggest that lysine catabolism is regulated primarily by the first enzyme LKR, while the excess level of SDH enables efficient flux of lysine catabolism following the LKR step. Analysis of transgenic plants expressing beta-glucoronidase fusion constructs with the AtLKR/SDH and monofunctional AtSDH promoters demonstrated that transcriptional regulation contributes to the modulation of expression of the bifunctional LKR/SDH and monofunctional SDH gene products in response to hormonal and metabolic signals. To test whether the enhanced expression of the LKR/SDH gene under various hormonal and metabolic signals is correlated with enhanced lysine catabolism, wild-type Arabidopsis and a knockout mutant lacking lysine catabolism were exposed to abscisic acid and sugar starvation. Free lysine accumulated to significantly higher levels in this knockout mutant than in the wild-type plants.

  19. The effect of fasting and refeeding on mRNA expression of PepT1 and gastrointestinal hormones regulating digestion and food intake in zebrafish (Danio rerio).

    Science.gov (United States)

    Koven, William; Schulte, Patricia

    2012-12-01

    In vertebrates, a significant part of ingested protein is absorbed as di- and tripeptides through a brush border membrane proton/oligopeptide transporter protein called PepT1. The aim of the present study was to determine the effect of short-term food deprivation and refeeding in adult zebrafish (Danio rerio) on gastrointestinal mRNA expression of PepT1 as well as on the satiety hormones cholecystokinin (CCK), gastrin-releasing peptide (GRP) and ghrelin (GHR) in order to elucidate a potential mechanism driving compensatory growth. Sixty adult zebrafish were stocked in a 40-L aquarium and fed daily a commercial flake diet to satiation for 10 days where the digestive tracts (DT) of sampled fish (n = 5) were dissected out. Samplings were repeated following 1, 2 and 5 days of food deprivation and after 1, 2 and 5 days of refeeding. The RNA was extracted from all sampled DTs and analyzed by quantitative real-time PCR for the mRNA expression of PepT1, rRNA 18S, CCK, GRP and GHR. PepT1 mRNA expression increased with successive refeedings reaching a level approximately 8 times higher than pre-fast levels. CCK, GRP and GHR mRNA levels also decreased during fasting, but increased only to pre-fasting levels with refeeding. Overall, the results suggest that PepT1 may be a contributing mechanism to compensatory growth that could influence CCK secretion and GRP and GHR activity.

  20. IL-2 induction of IL-1 beta mRNA expression in monocytes. Regulation by agents that block second messenger pathways

    DEFF Research Database (Denmark)

    Kovacs, E J; Brock, B; Varesio, L

    1989-01-01

    We have previously shown that in mixed cultures of PBL incubation with human rIL-2 induces the rapid expression of IL-1 alpha and IL-1 beta mRNA. Because studies have demonstrated that IL-2R can be expressed on the surface of human peripheral blood monocytes, we chose to investigate whether IL-1 ...

  1. AtLa1 protein initiates IRES-dependent translation of WUSCHEL mRNA and regulates the stem cell homeostasis of Arabidopsis in response to environmental hazards.

    Science.gov (United States)

    Cui, Yuchao; Rao, Shaofei; Chang, Beibei; Wang, Xiaoshuang; Zhang, Kaidian; Hou, Xueliang; Zhu, Xueyi; Wu, Haijun; Tian, Zhaoxia; Zhao, Zhong; Yang, Chengwei; Huang, Tao

    2015-10-01

    Plant stem cells are hypersensitive to environmental hazards throughout their life cycle, but the mechanism by which plants safeguard stem cell homeostasis in response to environmental hazards is largely unknown. The homeodomain transcription factor WUSCHEL (WUS) protein maintains the stem cell pool in the shoot apical meristem of Arabidopsis. Here, we demonstrate that the translation of WUS mRNA is directed by an internal ribosomal entry site (IRES) located in the 5'-untranslated region. The AtLa1 protein, an RNA-binding factor, binds to the 5'-untranslated region and initiates the IRES-dependent translation of WUS mRNA. Knockdown of AtLa1 expression represses the WUS IRES-dependent translation and leads to the arrest of growth and development. The AtLa1 protein is mainly located in the nucleoplasm. However, environmental hazards promote the nuclear-to-cytoplasmic translocation of the AtLa1 protein, which further enhances the IRES-dependent translation of WUS mRNA. Genetic evidence indicates that the WUS protein increases the tolerance of the shoot apical meristem to environmental hazards. Based on these results, we conclude that the stem cell niche in Arabidopsis copes with environmental hazards by enhancing the IRES-dependent translation of WUS mRNA under the control of the AtLa1 protein. © 2015 John Wiley & Sons Ltd.

  2. TREM-1 is a positive regulator of TNF-α and IL-8 production in U937 foam cells

    Directory of Open Access Journals (Sweden)

    Yu-Shi Wang

    2012-05-01

    Full Text Available The purpose of our study was to investigate the expression levels of TREM-1 (triggering receptor expressed on myeloid cells-1 in U937 foam cells and determine whether TREM-1 regulates the production of tumor necrosis factor-alpha and interleukin-8 in these cells.Human U937 cells were incubated with phorbol 12-myristate 13-acetate and then oxidized human low-density lipoprotein to induce foam cell formation. Oil red O staining was used to identify the foam cells. The production of IL-8 and TNF-α by U937 foam cells was assayed by enzyme-linked immunosorbent assay. The expression of TREM-1 mRNA in U937 foam cells was detected by reverse transcription-polymerase chain reaction. Moreover, U937 foam cells were transfected by small interfering RNA using Lipofectamine 2000 to knockdown TREM-1. Western blot was performed to assay protein expression of TREM-1 and ELISA was used to examine the effect of TREM-1 knockdown on IL-8 and TNF-α production.PMA and ox-LDL induced U937 cells to form foam cells. The production of TNF-α and IL-8 was found to be significantly elevated in U937 foam cells, concomitant with a significant up-regulation of TREM-1 mRNA. TREM-1 siRNA was able to partially silence the expression of TREM-1 protein and remarkably inhibited TNF-α and IL-8 production in U937 foam cells, suggesting that TREM-1 is a positive regulator of TNF-α and IL-8 production in U937 foam cells.Our finding that TREM-1 controls the production of IL-8 and TNF-α in U937 foam cells defines a potentially critical role of TREM-1 in the pathogenesis of atherosclerosis and implicates TREM-1 as a potential therapeutic target for the disease.

  3. Reduction of adhesion molecule production and alteration of eNOS and endothelin-1 mRNA expression in endothelium by Euphorbia hirta L. through its beneficial β-amyrin molecule.

    Science.gov (United States)

    Shih, Mei Fen; Cherng, Jong Yuh

    2014-07-18

    The inflammatory reaction in large blood vessels involves up-regulation of vascular adhesion molecules such as endothelial cell selectin (E-selectin), soluble vascular cell adhesion molecule (sVCAM)-1, and soluble intercellular adhesion molecule (sICAM)-1. These vascular dysfunctions are associated with the development of atherosclerosis. β-Amyrin, an active component of Euphorbia hirta L., has potent anti-inflammatory effects. So far, its preventive effects against the expression of inflammatory mediator-induced adhesion molecules have not been investigated. Endothelial cells (SVEC4-10 cell line) were treated with 50% RAW conditioned media (i.e., normal SVEC4-10 culture media contains 50% of lipopolysaccharide-activated macrophage culture media) without or with β-amyrin (0.6 and 0.3 µM). The production levels of E-selectin, sICAM-1, and sVCAM-1 in the SVEC4-10 cells were measured with ELISA assay kits. Under the same treatment conditions, expression of endothelin (ET)-1 and endothelial type of NO synthase (eNOS) mRNA were analyzed by RT-PCR and agarose gel. With β-amyrin, the 50% RAW conditioned media-induced E-selectin, sICAM-1, and sVCAM-1 levels as well as ET-1 gene expression were all suppressed. β-Amyrin treatment also restored the 50% RAW conditioned media-suppressed eNOS mRNA expression. These data indicate that β-amyrin is potentially useful in preventing chronic inflammation-related vascular diseases.

  4. Production of a Locus- and Allele-Specific Monoclonal Antibody for the Characterization of SLA-1*0401 mRNA and Protein Expression Levels in MHC-Defined Microminipigs.

    Directory of Open Access Journals (Sweden)

    Yoshie Kametani

    Full Text Available The class I major histocompatibility complex (MHC presents self-developed peptides to specific T cells to induce cytotoxity against infection. The MHC proteins are encoded by multiple loci that express numerous alleles to preserve the variability of the antigen-presenting ability in each species. The mechanism regulating MHC mRNA and protein expression at each locus is difficult to analyze because of the structural and sequence similarities between alleles. In this study, we examined the correlation between the mRNA and surface protein expression of swine leukocyte antigen (SLA-1*0401 after the stimulation of peripheral blood mononuclear cells (PBMCs by Staphylococcus aureus superantigen toxic shock syndrome toxin-1 (TSST-1. We prepared a monoclonal antibody (mAb against a domain composed of Y102, L103 and L109 in the α2 domain. The Hp-16.0 haplotype swine possess only SLA-1*0401, which has the mAb epitope, while other haplotypes possess 0 to 3 SLA classical class I loci with the mAb epitopes. When PBMCs from SLA-1*0401 homozygous pigs were stimulated, the SLA-1*0401 mRNA expression level increased until 24 hrs and decreased at 48 hrs. The kinetics of the interferon regulatory transcription factor-1 (IRF-1 mRNA level were similar to those of the SLA-1*0401 mRNA. However, the surface protein expression level continued to increase until 72 hrs. Similar results were observed in the Hp-10.0 pigs with three mAb epitopes. These results suggest that TSST-1 stimulation induced both mRNA and surface protein expression of class I SLA in the swine PBMCs differentially and that the surface protein level was sustained independently of mRNA regulation.

  5. Impact of Environmental Factors on the Regulation of Cyanotoxin Production

    Directory of Open Access Journals (Sweden)

    Thangavelu Boopathi

    2014-06-01

    Full Text Available Cyanobacteria are capable of thriving in almost all environments. Recent changes in climatic conditions due to increased human activities favor the occurrence and severity of harmful cyanobacterial bloom all over the world. Knowledge of the regulation of cyanotoxins by the various environmental factors is essential for effective management of toxic cyanobacterial bloom. In recent years, progress in the field of molecular mechanisms involved in cyanotoxin production has paved the way for assessing the role of various factors on the cyanotoxin production. In this review, we present an overview of the influence of various environmental factors on the production of major group of cyanotoxins, including microcystins, nodularin, cylindrospermopsin, anatoxins and saxitoxins.

  6. Effect of IFN-γ, IL-12 and IL-10 cytokine production and mRNA expression in tuberculosis patients with diabetes mellitus and their household contacts.

    Science.gov (United States)

    Meenakshi, Ponnana; Ramya, Sivangala; Lavanya, Joshi; Vijayalakshmi, Valluri; Sumanlatha, Gaddam

    2016-05-01

    The study was carried out to understand the influence of IFN-γ, IL-12 and IL-10 cytokine production and expression in tuberculosis patients with diabetes mellitus (TBDM) and their household contacts (HHC). The study involved a total of 300 subjects, 50 in each category of TBDM, TBDM HHC, pulmonary tuberculosis patients (PTB), PTB HHC, DM and healthy controls (HC). TBDM, PTB and their HHC, 25 each were followed at different intervals to determine their immune responses in Ag85A stimulated culture supernatants by Enzyme Linked Immunosorbent Assay (ELISA). mRNA expression by TRIZOL method in 5 cases of each category and follow-up studies were performed. IFN-γ and IL-12 cytokine production markedly decreased and that of IL-10 increased after Ag85A M.tb stimulation, however anti TB treatment reconstituted the response in TBDM and PTB patients. The household contacts revealed cytokine gene expression similar to that of patients and two of them developed the disease during follow-up. Cytokine responses of the patients retained after treatment highlighting the antigen importance, hence further studies with recombinant cytokines may help in coming up with a biomarker. Analogous immune responses of household contacts with the TBDM and PTB patients may assist in recognizing the high risk individuals. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Serotonin 2A and 2C receptor biosynthesis in the rodent striatum during postnatal development: mRNA expression and functional linkage to neuropeptide gene regulation.

    Science.gov (United States)

    Basura, G J; Walker, P D

    2000-11-01

    The present study was designed to determine if there are region-specific differences in serotonin (5-HT) neurotransmission and 5-HT receptor expression that may limit the stimulatory effects of the 5-HT releaser p-chloroamphetamine (pCA) on striatal neuropeptide gene expression to the posterior striatum (P-STR) during postnatal maturation. Sprague-Dawley rat brains from postnatal days (PND) 1-35 were processed for 5-HT(2A) and 5-HT(2C) receptor mRNA expression by in situ hybridization and monoamine analysis by HPLC. Within the P-STR, 5-HT(2A) receptor mRNA expression reached young adult (PND 35) levels by PND 3, while levels in the A-STR were significantly less (range: 1.43 +/- 0.219-6. 36 +/- 0.478) than P-STR (5.36 +/- 0.854-12.11 +/- 1.08) at each respective age throughout the time course. 5-HT(2C) receptor mRNA expression reached young adult levels at PND 7 in the A-STR and by PND 3 in the P-STR. At each PND age 5-HT(2C) receptor mRNA levels within the P-STR were significantly less (6.23 +/- 1.02-12.32 +/- 0.427) than the A-STR (7.31 +/- 1.65-26.84 +/- 2.24). 5-HT content increased across the developmental time course within the P-STR (5.01 +/- 0.327-15.7 +/- 1.03 ng/mg protein) and A-STR (2.97 +/- 0. 223-11.2 +/- 0.701 ng/mg protein). Four hours following injection (i. p.) of pCA (10 mg/kg), preprotachykinin (PPT) mRNA levels increased 89% in the P-STR but not the anterior (A-STR) striatum of the 3-week-old rat, which were prevented by preinjection (30 min, i.p.) of the 5-HT(2) receptor antagonist ritanserin (1 mg/kg). Together, these data suggest that faster maturity of 5-HT(2A) receptor expression in the P-STR may be sufficient to convey the region-specific acute stimulatory effects of pCA on PPT mRNA transcription in the developing rodent striatum. These results provide further evidence that the influence of 5-HT on neuropeptide gene expression is far stronger in caudal vs. rostral striatal regions during postnatal development. Copyright 2000 Wiley

  8. UCP2 mRNA expression is dependent on glucose metabolism in pancreatic islets

    Energy Technology Data Exchange (ETDEWEB)

    Dalgaard, Louise T., E-mail: ltd@ruc.dk [Division of Endocrinology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA (United States); Department of Science, Systems and Models, Roskilde University (Denmark)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer UCP2 mRNA levels are decreased in islets of Langerhans from glucokinase deficient mice. Black-Right-Pointing-Pointer UCP2 mRNA up-regulation by glucose is dependent on glucokinase. Black-Right-Pointing-Pointer Absence of UCP2 increases GSIS of glucokinase heterozygous pancreatic islets. Black-Right-Pointing-Pointer This may protect glucokinase deficient mice from hyperglycemic damages. -- Abstract: Uncoupling Protein 2 (UCP2) is expressed in the pancreatic {beta}-cell, where it partially uncouples the mitochondrial proton gradient, decreasing both ATP-production and glucose-stimulated insulin secretion (GSIS). Increased glucose levels up-regulate UCP2 mRNA and protein levels, but the mechanism for UCP2 up-regulation in response to increased glucose is unknown. The aim was to examine the effects of glucokinase (GK) deficiency on UCP2 mRNA levels and to characterize the interaction between UCP2 and GK with regard to glucose-stimulated insulin secretion in pancreatic islets. UCP2 mRNA expression was reduced in GK+/- islets and GK heterozygosity prevented glucose-induced up-regulation of islet UCP2 mRNA. In contrast to UCP2 protein function UCP2 mRNA regulation was not dependent on superoxide generation, but rather on products of glucose metabolism, because MnTBAP, a superoxide dismutase mimetic, did not prevent the glucose-induced up-regulation of UCP2. Glucose-stimulated insulin secretion was increased in UCP2-/- and GK+/- islets compared with GK+/- islets and UCP2 deficiency improved glucose tolerance of GK+/- mice. Accordingly, UCP2 deficiency increased ATP-levels of GK+/- mice. Thus, the compensatory down-regulation of UCP2 is involved in preserving the insulin secretory capacity of GK mutant mice and might also be implicated in limiting disease progression in MODY2 patients.

  9. Regulation of Adrenal Aldosterone Production by Serine Protease Prostasin

    Directory of Open Access Journals (Sweden)

    Takehiro Ko

    2010-01-01

    Full Text Available A serine protease prostasin has been demonstrated to have a pivotal role in the activation of the epithelial sodium channel. Systemic administration of adenovirus carrying human prostasin gene in rats resulted in an increase in plasma prostasin and aldosterone levels. However, the mechanism by which the elevation of prostasin levels in the systemic circulation stimulated the plasma aldosterone levels remains unknown. Therefore, we examined if prostasin increases the aldosterone synthesis in a human adrenocortical cell line (H295R cells. Luciferase assay using CYP11B2 promoter revealed that prostasin significantly increased the transcriptional activity of CYP11B2. Prostasin significantly increased both CYP11B2 mRNA expression and aldosterone production in a dose-dependent manner. Surprisingly, treatment with camostat mesilate, a potent prostasin inhibitor, had no effect on the aldosterone synthesis by prostasin and also a protease-dead mutant of prostasin significantly stimulated the aldosterone production. A T-type/L-type calcium channel blocker and a protein kinase C (PKC inhibitor significantly reduced the aldosterone synthesis by prostasin. Our findings suggest a stimulatory effect of prostasin on the aldosterone synthesis by adrenal gland through the nonproteolytic action and indicate a new role of prostasin in the systemic circulation.

  10. HIV-1 infection induces changes in expression of cellular splicing factors that regulate alternative viral splicing and virus production in macrophages

    Directory of Open Access Journals (Sweden)

    Purcell Damian FJ

    2008-02-01

    Full Text Available Abstract Background Macrophages are important targets and long-lived reservoirs of HIV-1, which are not cleared of infection by currently available treatments. In the primary monocyte-derived macrophage model of infection, replication is initially productive followed by a decline in virion output over ensuing weeks, coincident with a decrease in the levels of the essential viral transactivator protein Tat. We investigated two possible mechanisms in macrophages for regulation of viral replication, which appears to be primarily regulated at the level of tat mRNA: 1 differential mRNA stability, used by cells and some viruses for the rapid regulation of gene expression and 2 control of HIV-1 alternative splicing, which is essential for optimal viral replication. Results Following termination of transcription at increasing times after infection in macrophages, we found that tat mRNA did indeed decay more rapidly than rev or nef mRNA, but with similar kinetics throughout infection. In addition, tat mRNA decayed at least as rapidly in peripheral blood lymphocytes. Expression of cellular splicing factors in uninfected and infected macrophage cultures from the same donor showed an inverse pattern over time between enhancing factors (members of the SR family of RNA binding proteins and inhibitory factors (members of the hnRNP family. While levels of the SR protein SC35 were greatly up-regulated in the first week or two after infection, hnRNPs of the A/B and H groups were down-regulated. Around the peak of virus production in each culture, SC35 expression declined to levels in uninfected cells or lower, while the hnRNPs increased to control levels or above. We also found evidence for increased cytoplasmic expression of SC35 following long-term infection. Conclusion While no evidence of differential regulation of tat mRNA decay was found in macrophages following HIV-1 infection, changes in the balance of cellular splicing factors which regulate alternative

  11. The Canadian Natural Health Products (NHP) regulations: industry compliance motivations.

    Science.gov (United States)

    Laeeque, Hina; Boon, Heather; Kachan, Natasha; Cohen, Jillian Clare; D'Cruz, Joseph

    2007-06-01

    This qualitative study explores corporations' motivations to comply with new natural health products (NHP) Regulations in Canada. Interviews were conducted with representatives from 20 Canadian NHP companies. Findings show that the rationale for compliance differs for large compared to small and medium-sized enterprises (SMEs). Large firms are motivated to comply with the regulations because of the deterrent fear of negative media coverage, social motivations, ability to comply and maintaining a competitive market advantage. In contrast, SMEs are motivated to comply due to the deterrent fear of legal prosecution and a sense of duty.

  12. The Canadian Natural Health Products (NHP Regulations: Industry Compliance Motivations

    Directory of Open Access Journals (Sweden)

    Hina Laeeque

    2007-01-01

    Full Text Available This qualitative study explores corporations' motivations to comply with new natural health products (NHP Regulations in Canada. Interviews were conducted with representatives from 20 Canadian NHP companies. Findings show that the rationale for compliance differs for large compared to small and medium-sized enterprises (SMEs. Large firms are motivated to comply with the regulations because of the deterrent fear of negative media coverage, social motivations, ability to comply and maintaining a competitive market advantage. In contrast, SMEs are motivated to comply due to the deterrent fear of legal prosecution and a sense of duty.

  13. 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 ei......) 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......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...

  14. NFAT regulates calcium-sensing receptor-mediated TNF production

    Energy Technology Data Exchange (ETDEWEB)

    abdullah, huda ismail; Pedraza, Paulina L.; Hao, Shoujin; Rodland, Karin D.; McGiff, John C.; Ferreri, Nicholas R.

    2006-05-01

    Because nuclear factor of activated T cells (NFAT) has been implicated in TNF production as well as osmoregulation and salt and water homeostasis, we addressed whether calcium-sensing receptor (CaR)-mediated TNF production in medullary thick ascending limb (mTAL) cells was NFAT dependent. TNF production in response to addition of extracellular Ca2+ (1.2 mM) was abolished in mTAL cells transiently transfected with a dominant-negative CaR construct (R796W) or pretreated with the phosphatidylinositol phospholipase C (PI-PLC) inhibitor U-73122. Cyclosporine A (CsA), an inhibitor of the serine/threonine phosphatase calcineurin, and a peptide ligand, VIVIT, that selectively inhibits calcineurin-NFAT signaling, also prevented CaR-mediated TNF production. Increases in calcineurin activity in cells challenged with Ca2+ were inhibited after pretreatment with U-73122 and CsA, suggesting that CaR activation increases calcineurin activity in a PI-PLC-dependent manner. Moreover, U-73122, CsA, and VIVIT inhibited CaR-dependent activity of an NFAT construct that drives expression of firefly luciferase in transiently transfected mTAL cells. Collectively, these data verify the role of calcineurin and NFAT in CaR-mediated TNF production by mTAL cells. Activation of the CaR also increased the binding of NFAT to a consensus oligonucleotide, an effect that was blocked by U-73122 and CsA, suggesting that a calcineurin- and NFAT-dependent pathway increases TNF production in mTAL cells. This mechanism likely regulates TNF gene transcription as U-73122, CsA, and VIVIT blocked CaR-dependent activity of a TNF promoter construct. Elucidating CaR-mediated signaling pathways that regulate TNF production in the mTAL will be crucial to understanding mechanisms that regulate extracellular fluid volume and salt balance.

  15. Regulation of aromatic alcohol production in Candida albicans.

    Science.gov (United States)

    Ghosh, Suman; Kebaara, Bessie W; Atkin, Audrey L; Nickerson, Kenneth W

    2008-12-01

    Colonization by the fungal pathogen Candida albicans varies significantly, depending upon the pH and availability of oxygen. Because of our interest in extracellular molecules as potential quorum-sensing molecules, we examined the physiological conditions which regulate the production of the aromatic alcohols, i.e., phenethyl alcohol, tyrosol, and tryptophol. The production of these fusel oils has been well studied for Saccharomyces cerevisiae. Our data show that aromatic alcohol yields for C. albicans are determined by growth conditions. These conditions include the availability of aromatic amino acids, the pH, oxygen levels, and the presence of ammonium salts. For example, for wild-type C. albicans, tyrosol production varied 16-fold merely with the inclusion of tyrosine or ammonium salts in the growth medium. Aromatic alcohol production also depends on the transcription regulator Aro80p. Our results are consistent with aromatic alcohol production-aromatic transaminases (gene products for ARO8 and ARO9), aromatic decarboxylase (ARO10), and alcohol dehydrogenase (ADH)-via the fusel oil pathway. The expression of ARO8, ARO9, and ARO10 is also pH dependent. ARO8 and ARO9 were alkaline upregulated, while ARO10 was alkaline downregulated. The alkaline-dependent change in expression of ARO8 was Rim101 independent, while the expression of ARO9 was Rim101 dependent.

  16. Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA

    Energy Technology Data Exchange (ETDEWEB)

    Milsted, A.; Barna, B.P.; Ransohoff, R.M.; Brosnihan, K.B.; Ferrario, C.M. (Cleveland Clinic Foundation, OH (USA))

    1990-08-01

    The authors have identified human cultured cell lines that are useful for studying angiotensinogen gene expression and its regulation in the central nervous system. A model cell system of human central nervous system origin expressing angiotensinogen has not previously been available. Expression of angiotensinogen mRNA appears to be a basal property of noninduced human astrocytes, since astrocytic cell lines derived from human glioblastomas or nonneoplastic human brain tissue invariably produced angiotensinogen mRNA. In situ hybridization histochemistry revealed that angiotensinogen mRNA production was not limited to a subpopulation of astrocytes because >99% of cells in these cultures contained angiotensinogen mRNA. These cell lines will be useful in studies of the molecular mechanisms controlling angiotensin synthesis and the role of biologically active angiotensin in the human brain by allowing the authors to examine regulation of expression of the renin-angiotensin system in human astrocyte cultures.

  17. Biosynthesis and regulation of cyanogenic glycoside production in forage plants.

    Science.gov (United States)

    Sun, Zhanmin; Zhang, Kaixuan; Chen, Cheng; Wu, Yanmin; Tang, Yixiong; Georgiev, Milen I; Zhang, Xinquan; Lin, Min; Zhou, Meiliang

    2017-10-12

    The natural products cyanogenic glycosides (CNglcs) are present in various forage plant species including Sorghum spp., Trifolium spp., and Lotus spp. The release of toxic hydrogen cyanide (HCN) from endogenous CNglcs, which is known as cyanogenesis, leads to a serious problem for animal consumption while as defensive secondary metabolites, CNglcs play multiple roles in plant development and responses to adverse environment. Therefore, it is highly important to fully uncover the molecular mechanisms of CNglc biosynthesis and regulation to manipulate the contents of CNglcs in forage plants for fine-tuning the balance between defensive responses and food safety. This review summarizes recent studies on the production, function, polymorphism, and regulation of CNglcs in forage plants, aiming to provide updated knowledge on the ways to manipulate CNglcs for further beneficial economic effects.

  18. Regulation of phytotoxin production in Pseudomonas syringae pv. tabaci

    Energy Technology Data Exchange (ETDEWEB)

    Weaver, L.M.; Ghosh, S.; Knight, T.J.; Unkefer, P.J. (Los Alamos National Lab., NM (United States))

    1991-05-01

    Pseudomonas syringae pv. tabaci, a pathogen of tobacco, is capable of colonizing the rhizosphere of many plants. This pathogen excretes tabtoxinine-{beta}-lactam (T{beta}L), an active site directed, irreversible inhibitor of glutamine synthetase. T{beta}L is produced in planta, in the rhizosphere, and under certain culture conditions. However, the factors which regulated T{beta}L production in these environments are unknown. As a first step in characterizing T{beta}L synthesis by P. syringae pv. tabaci, the authors have determined the effects of root exudates and various nutrients on production of T{beta}L by P. syringae pv. tabaci PT113.

  19. TNFα PRODUCTION AND APOPTOSIS REGULATION IN VIRAL HEPATITIS TYPE C

    Directory of Open Access Journals (Sweden)

    V. V. Novitsky

    2005-01-01

    Full Text Available Abstract. Chronical course of infection caused by hepatitis C virus is accompanied by increase Fas-positive lymphocytes of peripheral blood. Cultivation of agglutinin-stimulated mononuclear blood cells of patients with chronic hepatitis C revealed inhibition of apoptotic reactions of blood lymphocytes. This fact correlated with decrease in production of TNFα and accelerated synthesis of soluble receptor for this cytokine. We suggest a virus-specific influence on apoptosis regulation of target cells.

  20. Best practices in regulation of blood and blood products.

    Science.gov (United States)

    Epstein, Jay S

    2012-05-01

    The need for blood regulation arises from the inherent risks of blood transfusion, which are minimized through implementation of standards. Regulatory oversight is advocated by the World Health Organization (WHO) as an essential element of any blood system to ensure such standards are met. The WHO Blood Regulators Network has developed "Assessment Criteria for National Blood Regulatory Systems" that describe the legal authority and functions of a fully competent blood regulator. The core functions include licensing and/or registration of blood establishments, marketing approval of blood products, oversight of all associated substances and devices, control of clinical trials, access to an independent laboratory for product assessments, lot release, and hemovigilance systems. Regulatory policy-making for blood safety is needed to address emerging threats, to consider the risks and benefits of new products and technologies, and to respond to adverse events. Structured policy-making processes are essential to ensure that decisions are science-based, with appropriate consideration of relevant economic and social factors. Decision making is especially challenging in situations of scientific uncertainty, where prudent precautionary measures may be appropriate based on assessments of risk and feasibility of meaningful interventions. There is international interest in finding a common framework for addressing blood safety decisions. Published by Elsevier Ltd.

  1. One Gene and Two Proteins: a Leaderless mRNA Supports the Translation of a Shorter Form of the Shigella VirF Regulator

    Directory of Open Access Journals (Sweden)

    Maria Letizia Di Martino

    2016-11-01

    Full Text Available VirF, an AraC-like activator, is required to trigger a regulatory cascade that initiates the invasive program of Shigella spp., the etiological agents of bacillary dysentery in humans. VirF expression is activated upon entry into the host and depends on many environmental signals. Here, we show that the virF mRNA is translated into two proteins, the major form, VirF30 (30 kDa, and the shorter VirF21 (21 kDa, lacking the N-terminal segment. By site-specific mutagenesis and toeprint analysis, we identified the translation start sites of VirF30 and VirF21 and showed that the two different forms of VirF arise from differential translation. Interestingly, in vitro and in vivo translation experiments showed that VirF21 is also translated from a leaderless mRNA (llmRNA whose 5′ end is at position +309/+310, only 1 or 2 nucleotides upstream of the ATG84 start codon of VirF21. The llmRNA is transcribed from a gene-internal promoter, which we identified here. Functional analysis revealed that while VirF30 is responsible for activation of the virulence system, VirF21 negatively autoregulates virF expression itself. Since VirF21 modulates the intracellular VirF levels, this suggests that transcription of the llmRNA might occur when the onset of the virulence program is not required. We speculate that environmental cues, like stress conditions, may promote changes in virF mRNA transcription and preferential translation of llmRNA.

  2. Guanylin and uroguanylin mRNA expression is increased following Roux-en-Y gastric bypass, but guanylins do not play a significant role in body weight regulation and glycemic control

    DEFF Research Database (Denmark)

    Fernandez-Cachon, María L; Pedersen, Søren L; Rigbolt, Kristoffer T

    2017-01-01

    for potentially contributing to the beneficial metabolic effects of RYGB. METHODS: Enteroendocrine cells were harvested peri- and post-RYGB, and GUCA2A/GUCA2B mRNA expression was compared. GN, UGN and their prohormones (proGN, proUGN) were administered subcutaneously in normal-weight mice to evaluate effects......: GN and UGN, as well as their prohormones, do not seem to play a significant role in body weight regulation and glycemic control, suggesting that guanylin-family peptides do not show promise as targets for the treatment of obesity or diabetes....

  3. Role of oxygen in the regulation of Leydig tumor derived MA-10 cell steroid production: the effect of cobalt chloride.

    Science.gov (United States)

    Kumar, Anand; Rani, Lata; Dhole, Bodhana

    2014-04-01

    We have earlier shown that cobalt chloride (CoCl2)-induced hypoxia and second messenger 8-bromoadenosine 3', 5'-cyclic adenosine monophosphate (8-Br-cAMP) stimulates vascular endothelial growth factor (VEGF) production in Leydig tumor cell derived MA-10 cells. Both stimuli follow common signal transduction pathways including protein kinase A (PK-A), extracellular regulated kinase 1/2 (ERK1/2), and phosphatidyl inositol-3 kinase/akt (PI3-K/Akt) pathways in the stimulation of VEGF by MA-10 cells. In the present study we investigated the role of CoCl2 and 8-Br-cAMP on steroid production in MA-10 cells. The MA-10 cells were cultured in Waymouth MB 752/1 medium, supplemented with 15% heat inactivated horse serum. Progesterone was estimated by radioimmunoassay (RIA).We report that 8-Br-cAMP stimulated progesterone production by the MA-10 cells whereas CoCl2 inhibited the same. Also, 8-Br-cAMP stimulated steroidogenic acute regulatory protein (StAR) and cytochrome P450 side-chain cleavage enzyme (P450scc) mRNAs expression. However, CoCl2 had no effect on StAR mRNA. Cobalt chloride directly inhibited the expression of P450scc mRNA. The decrease in progesterone production could be attributed to three different mechanisms, (1) an increase in production of reactive oxygen species (ROS), (2) an increase in HIF-1α activity, and (3) ultimately a decrease in the level of cytochrome P450 side chain cleavage (CYT P450scc). Hypoxia has an action and mechanism of action similar to that of gonadotropins on VEGF production, whereas they have a contrasting effect on steroidogenesis. This study suggests that hypoxia could be as important as gonadotropins in regulating Leydig cell steroidogenesis.

  4. Secreted Thrombospondin-1 Regulates Macrophage Interleukin-1β Production and Activation through CD47.

    Science.gov (United States)

    Stein, Erica V; Miller, Thomas W; Ivins-O'Keefe, Kelly; Kaur, Sukhbir; Roberts, David D

    2016-01-27

    Thrombospondin-1 regulates inflammation by engaging several cell surface receptors and by modulating activities of other secreted factors. We have uncovered a novel role of thrombospondin-1 in modulating production and activation of the proinflammatory cytokine IL-1β by human and murine macrophages. Physiological concentrations of thrombospondin-1 limit the induction by lipopolysaccharide of IL-1β mRNA and total protein production by human macrophages. This inhibition can be explained by the ability of thrombospondin-1 to disrupt the interaction between CD47 and CD14, thereby limiting activation of NFκB/AP-1 by lipopolysaccharide. Only the CD47-binding domain of thrombospondin-1 exhibits this activity. In contrast, CD47, CD36, and integrin-binding domains of thrombospondin-1 independently enhance the inflammasome-dependent maturation of IL-1β in human THP-1 monocyte-derived macrophages. Correspondingly, mouse bone marrow-derived macrophages that lack either thrombospondin-1 or CD47 exhibit diminished induction of mature IL-1β in response to lipopolysaccharide. Lack of CD47 also limits lipopolysaccharide induction of IL-1β, NLRP3, and caspase-1 mRNAs. These data demonstrate that thrombospondin-1 exerts CD47-dependent and -independent pro-and anti-inflammatory effects on the IL-1β pathway. Therefore, thrombospondin-1 and its receptor CD47 may be useful targets for limiting the pro-inflammatory effects of lipopolysaccharide and for treating endotoxemia.

  5. HuD and the Survival Motor Neuron Protein Interact in Motoneurons and Are Essential for Motoneuron Development, Function, and mRNA Regulation.

    Science.gov (United States)

    Hao le, Thi; Duy, Phan Q; An, Min; Talbot, Jared; Iyer, Chitra C; Wolman, Marc; Beattie, Christine E

    2017-11-29

    Motoneurons establish a critical link between the CNS and muscles. If motoneurons do not develop correctly, they cannot form the required connections, resulting in movement defects or paralysis. Compromised development can also lead to degeneration because the motoneuron is not set up to function properly. Little is known, however, regarding the mechanisms that control vertebrate motoneuron development, particularly the later stages of axon branch and dendrite formation. The motoneuron disease spinal muscular atrophy (SMA) is caused by low levels of the survival motor neuron (SMN) protein leading to defects in vertebrate motoneuron development and synapse formation. Here we show using zebrafish as a model system that SMN interacts with the RNA binding protein (RBP) HuD in motoneurons in vivo during formation of axonal branches and dendrites. To determine the function of HuD in motoneurons, we generated zebrafish HuD mutants and found that they exhibited decreased motor axon branches, dramatically fewer dendrites, and movement defects. These same phenotypes are present in animals expressing low levels of SMN, indicating that both proteins function in motoneuron development. HuD binds and transports mRNAs and one of its target mRNAs, Gap43, is involved in axonal outgrowth. We found that Gap43 was decreased in both HuD and SMN mutants. Importantly, transgenic expression of HuD in motoneurons of SMN mutants rescued the motoneuron defects, the movement defects, and Gap43 mRNA levels. These data support that the interaction between SMN and HuD is critical for motoneuron development and point to a role for RBPs in SMA.SIGNIFICANCE STATEMENT In zebrafish models of the motoneuron disease spinal muscular atrophy (SMA), motor axons fail to form the normal extent of axonal branches and dendrites leading to decreased motor function. SMA is caused by low levels of the survival motor neuron (SMN) protein. We show in motoneurons in vivo that SMN interacts with the RNA binding

  6. Insulin immuno-neutralization decreases food intake in chickens without altering hypothalamic mRNA levels for genes involved in regulation of food intake and metabolism

    Science.gov (United States)

    Chickens are characterized by rather unique glucose homeostasis, with relatively high blood glucose levels, reduced glucose sensitivity of pancreatic cells, and large resistance to exogenous insulin. In mammals, insulin regulates blood glucose level but also plays a key role in appetite regulation ...

  7. Thyroid hormone negatively regulates CDX2 and SOAT2 mRNA expression via induction of miRNA-181d in hepatic cells

    Energy Technology Data Exchange (ETDEWEB)

    Yap, Chui Sun; Sinha, Rohit Anthony [Cardiovascular and Metabolic Disorders, Duke-NUS Graduate Medical School, 8, College Road, Singapore 169857 (Singapore); Ota, Sho; Katsuki, Masahito [Department of Molecular Endocrinology, Tohoku University Graduate School of Medicine, Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan); Yen, Paul Michael, E-mail: paul.yen@duke-nus.edu.sg [Cardiovascular and Metabolic Disorders, Duke-NUS Graduate Medical School, 8, College Road, Singapore 169857 (Singapore)

    2013-11-01

    Highlights: •Thyroid hormone induces miR-181d expression in human hepatic cells and mouse livers. •Thyroid hormone downregulates CDX2 and SOAT2 (or ACAT2) via miR-181d. •miR-181d reduces cholesterol output from human hepatic cells. -- Abstract: Thyroid hormones (THs) regulate transcription of many metabolic genes in the liver through its nuclear receptors (TRs). Although the molecular mechanisms for positive regulation of hepatic genes by TH are well understood, much less is known about TH-mediated negative regulation. Recently, several nuclear hormone receptors were shown to downregulate gene expression via miRNAs. To further examine the potential role of miRNAs in TH-mediated negative regulation, we used a miRNA microarray to identify miRNAs that were directly regulated by TH in a human hepatic cell line. In our screen, we discovered that miRNA-181d is a novel hepatic miRNA that was regulated by TH in hepatic cell culture and in vivo. Furthermore, we identified and characterized two novel TH-regulated target genes that were downstream of miR-181d signaling: caudal type homeobox 2 (CDX2) and sterol O-acyltransferase 2 (SOAT2 or ACAT2). CDX2, a known positive regulator of hepatocyte differentiation, was regulated by miR-181d and directly activated SOAT2 gene expression. Since SOAT2 is an enzyme that generates cholesteryl esters that are packaged into lipoproteins, our results suggest miR-181d plays a significant role in the negative regulation of key metabolic genes by TH in the liver.

  8. Galanin-like peptide (GALP) neurone-specific phosphoinositide 3-kinase signalling regulates GALP mRNA levels in the hypothalamus of males and luteinising hormone levels in both sexes.

    Science.gov (United States)

    Aziz, R; Beymer, M; Negrón, A L; Newshan, A; Yu, G; Rosati, B; McKinnon, D; Fukuda, M; Lin, R Z; Mayer, C; Boehm, U; Acosta-Martínez, M

    2014-07-01

    Galanin-like peptide (GALP) neurones participate in the metabolic control of reproduction and are targets of insulin and leptin regulation. Phosphoinositide 3-kinase (PI3K) is common to the signalling pathways utilised by both insulin and leptin. Therefore, we investigated whether PI3K signalling in neurones expressing GALP plays a role in the transcriptional regulation of the GALP gene and in the metabolic control of luteinising hormone (LH) release. Accordingly, we deleted PI3K catalytic subunits p110α and p110β via conditional gene targeting (cKO) in mice (GALP-p110α/β cKO). To monitor PI3K signalling in GALP neurones, these animals were also crossed with Cre-dependent FoxO1GFP reporter mice. Compared to insulin-infused control animals, the PI3K-Akt-dependent FoxO1GFP nuclear exclusion in GALP neurones was abolished in GALP-p110α/β cKO mice. We next used food deprivation to investigate whether the GALP-neurone specific ablation of PI3K activity affected the susceptibility of the gonadotrophic axis to negative energy balance. Treatment did not affect LH levels in either sex. However, a significant genotype effect on LH levels was observed in females. By contrast, no genotype effect on LH levels was observed in males. A sex-specific genotype effect on hypothalamic GALP mRNA was observed, with fed and fasted GALP-p110α/β cKO males having lower GALP mRNA expression compared to wild-type fed males. Finally, the effects of gonadectomy and steroid hormone replacement on GALP mRNA levels were investigated. Compared to vehicle-treated mice, steroid hormone replacement reduced mediobasal hypothalamus GALP expression in wild-type and GALP-p110α/β cKO animals. In addition, within the castrated and vehicle-treated group and compared to wild-type mice, LH levels were lower in GALP-p110α/β cKO males. Double immunofluorescence using GALP-Cre/R26-YFP mice showed androgen and oestrogen receptor co-localisation within GALP neurones. Our data demonstrate that GALP

  9. G-Quadruplex in the NRF2 mRNA 5' Untranslated Region Regulates De Novo NRF2 Protein Translation under Oxidative Stress.

    Science.gov (United States)

    Lee, Sang C; Zhang, Jack; Strom, Josh; Yang, Danzhou; Dinh, Thai Nho; Kappeler, Kyle; Chen, Qin M

    2017-01-01

    Inhibition of protein synthesis serves as a general measure of cellular consequences of chemical stress. A few proteins are translated selectively and influence cell fate. How these proteins can bypass the general control of translation remains unknown. We found that low to mild doses of oxidants induce de novo translation of the NRF2 protein. Here we demonstrate the presence of a G-quadruplex structure in the 5' untranslated region (UTR) of NRF2 mRNA, as measured by circular dichroism, nuclear magnetic resonance, and dimethylsulfate footprinting analyses. Such a structure is important for 5'-UTR activity, since its removal by sequence mutation eliminated H2O2-induced activation of the NRF2 5' UTR. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics revealed elongation factor 1 alpha (EF1a) as a protein binding to the G-quadruplex sequence. Cells responded to H2O2 treatment by increasing the EF1a protein association with NRF2 mRNA, as measured by RNA-protein interaction assays. The EF1a interaction with small and large subunits of ribosomes did not appear to change due to H2O2 treatment, nor did posttranslational modifications, as measured by two-dimensional (2-D) Western blot analysis. Since NRF2 encodes a transcription factor essential for protection against tissue injury, our data have revealed a novel mechanism of cellular defense involving de novo NRF2 protein translation governed by the EF1a interaction with the G-quadruplex in the NRF2 5' UTR during oxidative stress. Copyright © 2016 American Society for Microbiology.

  10. Up-regulation of serotonin receptor 2B mRNA and protein in the peri-infarcted area of aged rats and stroke patients

    Science.gov (United States)

    Bădescu, George Mihai; Bogdan, Catalin; Weston, Ria; Slevin, Mark; Di Napoli, Mario; Popa-Wagner, Aurel

    2016-01-01

    Despite the fact that a high proportion of elderly stroke patients develop mood disorders, the mechanisms underlying late-onset neuropsychiatric and neurocognitive symptoms have so far received little attention in the field of neurobiology. In rodents, aged animals display depressive symptoms following stroke, whereas young animals recover fairly well. This finding has prompted us to investigate the expression of serotonin receptors 2A and 2B, which are directly linked to depression, in the brains of aged and young rats following stroke. Although the development of the infarct was more rapid in aged rats in the first 3 days after stroke, by day 14 the cortical infarcts were similar in size in both age groups i.e. 45% of total cortical volume in young rats and 55.7% in aged rats. We also found that the expression of serotonin receptor type B mRNA was markedly increased in the perilesional area of aged rats as compared to the younger counterparts. Furthermore, histologically, HTR2B protein expression in degenerating neurons was closely associated with activated microglia both in aged rats and human subjects. Treatment with fluoxetine attenuated the expression of Htr2B mRNA, stimulated post-stroke neurogenesis in the subventricular zone and was associated with an improved anhedonic behavior and an increased activity in the forced swim test in aged animals. We hypothesize that HTR2B expression in the infarcted territory may render degenerating neurons susceptible to attack by activated microglia and thus aggravate the consequences of stroke. PMID:27013593

  11. Anorexigenic and Orexigenic Hormone Modulation of Mammalian Target of Rapamycin Complex 1 Activity and the Regulation of Hypothalamic Agouti-Related Protein mRNA Expression

    Directory of Open Access Journals (Sweden)

    Kenneth R. Watterson

    2012-03-01

    Full Text Available Activation of mammalian target of rapamycin 1 (mTORC1 by nutrients, insulin and leptin leads to appetite suppression (anorexia. Contrastingly, increased AMP-activated protein kinase (AMPK activity by ghrelin promotes appetite (orexia. However, the interplay between these mechanisms remains poorly defined. The relationship between the anorexigenic hormones, insulin and leptin, and the orexigenic hormone, ghrelin, on mTORC1 signalling was examined using S6 kinase phosphorylation as a marker for changes in mTORC1 activity in mouse hypothalamic GT1-7 cells. Additionally, the contribution of AMPK and mTORC1 signalling in relation to insulin-, leptin- and ghrelin-driven alterations to mouse hypothalamic agouti-related protein (AgRP mRNA levels was examined. Insulin and leptin increase mTORC1 activity in a phosphoinositide-3-kinase (PI3K- and protein kinase B (PKB-dependent manner, compared to vehicle controls, whereas increasing AMPK activity inhibits mTORC1 activity and blocks the actions of the anorexigenic hormones. Ghrelin mediates an AMPK-dependent decrease in mTORC1 activity and increases hypothalamic AgRP mRNA levels, the latter effect being prevented by insulin in an mTORC1-dependent manner. In conclusion, mTORC1 acts as an integration node in hypothalamic neurons for hormone-derived PI3K and AMPK signalling and mediates at least part of the assimilated output of anorexigenic and orexigenic hormone actions in the hypothalamus.

  12. Transforming growth factor-beta 1 (TGF-beta1) promotes IL-2 mRNA expression through the up-regulation of NF-kappaB, AP-1 and NF-AT in EL4 cells.

    Science.gov (United States)

    Han, S H; Yea, S S; Jeon, Y J; Yang, K H; Kaminski, N E

    1998-12-01

    Transforming growth factor beta1 (TGF-beta1) has been previously shown to modulate interleukin 2 (IL-2) secretion by activated T-cells. In the present studies, we determined that TGF-beta1 induced IL-2 mRNA expression in the murine T-cell line EL4, in the absence of other stimuli. IL-2 mRNA expression was significantly induced by TGF-beta1 (0.1-1 ng/ml) over a relatively narrow concentration range, which led to the induction of IL-2 secretion. Under identical condition, we examined the effect of TGF-beta1 on the activity of nuclear factor AT (NF-AT), nuclear factor kappaB (NF-kappaB), activator protein-1 (AP-1) and octamer, all of which contribute to the regulation of IL-2 gene expression. Electrophoretic mobility shift assays showed that TGF-beta1 markedly increased NF-AT, NF-kappaB and AP-1 binding to their respective cognate DNA binding sites, whereas octamer binding remained constant, as compared with untreated cells. Employing a reporter gene expression system with p(NF-kappaB)3-CAT, p(NF-AT)3-CAT and p(AP-1)3-CAT, TGF-beta1 treatment of transfected EL4 cells induced a dose-related increase in chloramphenicol acetyltransferase activity that correlated well with the DNA binding profile found in the electrophoretic mobility shift assay studies. These results show that TGF-beta1, in the absence of any additional stimuli, up-regulates the activity of key transcription factors involved in IL-2 gene expression, including NF-AT, NF-kappaB and AP-1, to help promote IL-2 mRNA expression by EL4 cells.

  13. Neuropeptide Y (NPY), cocaine- and amphetamine-regulated transcript (CART) and cholecystokinin (CCK) in winter skate (Raja ocellata): cDNA cloning, tissue distribution and mRNA expression responses to fasting.

    Science.gov (United States)

    MacDonald, Erin; Volkoff, Hélène

    2009-04-01

    cDNAs encoding for neuropeptide Y (NPY), cocaine- and amphetamine-regulated transcript (CART) and cholecystokinin (CCK) were cloned in an elasmobranch fish, the winter skate. mRNA tissue distribution was examined for the three peptides as well as the effects of two weeks of fasting on their expression. Skate NPY, CART and CCK sequences display similarities with sequences for teleost fish but in general the degree of identity is relatively low (50%). All three peptides are present in brain and in several peripheral tissues, including gut and gonads. Within the brain, the three peptides are expressed in the hypothalamus, telencephalon, optic tectum and cerebellum. Two weeks of fasting induced an increase in telencephalon NPY and an increase in CCK in the gut but had no effects on hypothalamic NPY, CART and CCK, or on telencephalon CART. Our results provide basis for further investigation into the regulation of feeding in winter skate.

  14. IMMUNE REGULATING ES-PRODUCTS IN PARASITIC NEMATODES

    DEFF Research Database (Denmark)

    Bahlool, Qusay Zuhair Mohammad; Buchmann, Kurt; Kania, Per Walter

    Excretory/secretory (ES) products are molecules including various proteins produced by parasitic nematodes including larval A. simplex which is occurring in numerous marine fish hosts. The function of these substances and their effect on host physiology has not been fully described. The present...... work elucidates the effect of ES substances on the fish immune system by measuring immune gene expression in spleen and liver of rainbow trout (Oncorhynchus mykiss) injected intraperitoneally with ES products isolated from A. simplex third stage larvae. The overall gene expression profile of exposed...... fish showed a generalized down-regulation of the immune genes tested, suggesting a role of ES proteins in minimizing the immune reaction of rainbow trout against invading nematodes. We also tested the enzymatic activity of the ES proteins and found that lipase, esterase lipase, valine and cysteine...

  15. Gonadotropin regulation of in vitro androgen production by reptilian testes.

    Science.gov (United States)

    Wo Tsui, H; Licht, P

    1977-04-01

    The hormonal regulation of in vitro androgen production by minced testes from 7 species of reptiles representing the 3 major orders was studied using purified follicle stimulating hormone (FSH) and luteinizing hormone (LH) from tetrapod species. Androgen content was measured by radioimmunoassay. All 7 species showed a dose-dependent response to all preparations of FSH and LH tested. However, variations were found depending on the species tested and the source of the hormone. All snake hormones were particularly inactive in turtles. Some of the variation in relative potencies of hormone reflect phylogenetic specificity in the testes. Synergism between FSH and LH was tested in the sea turtle. While subliminal doses of FSH and LH produced a small stimulation of androgen production, each alone was ineffective. Both gonadotropins have intrinsic activity with regard to the stimulation of steroidogenesis.

  16. Integrated analysis of microRNA and mRNA expression and association with HIF binding reveals the complexity of microRNA expression regulation under hypoxia

    National Research Council Canada - National Science Library

    Camps, Carme; Saini, Harpreet K; Mole, David R; Choudhry, Hani; Reczko, Martin; Guerra-Assunção, José Afonso; Tian, Ya-Min; Buffa, Francesca M; Harris, Adrian L; Hatzigeorgiou, Artemis G; Enright, Anton J; Ragoussis, Jiannis

    2014-01-01

    .... Our aim is to investigate in depth the regulation of microRNA expression by hypoxia in the breast cancer cell line MCF-7, establish the relationship between microRNA expression and HIF binding sites...

  17. Klotho expression in long bones regulates FGF23 production during renal failure.

    Science.gov (United States)

    Kaludjerovic, Jovana; Komaba, Hirotaka; Sato, Tadatoshi; Erben, Reinhold G; Baron, Roland; Olauson, Hannes; Larsson, Tobias E; Lanske, Beate

    2017-05-01

    Circulating levels of bone-derived fibroblast growth factor 23 (FGF23) increase early during acute and chronic kidney disease and are associated with adverse outcomes. Membrane-bound Klotho acts as a permissive coreceptor for FGF23, and its expression was recently found in osteoblasts/osteocytes. We hypothesized that Klotho in bone cells is part of an autocrine feedback loop that regulates FGF23 expression during renal failure. Thus, we induced renal failure in mice with targeted deletion of Klotho in long bones. Uremic wild-type (KLfl/fl ) and knockout (Prx1-Cre;KLfl/fl ) mice both responded with reduced body weight, kidney atrophy, hyperphosphatemia, and increased bone turnover. Importantly, long bones of Prx1-Cre;KLfl/fl mice but not their axial skeleton failed to increase FGF23 expression as observed in uremic KLfl/fl mice. Consequently, Prx1-Cre;KLfl/fl mice had significantly lower serum FGF23 and parathyroid hormone levels, and higher renal 1-α-hydroxylase expression, serum 1,25-dihydroxyvitamin D, and calcium levels than KLfl/fl mice. These results were confirmed in two independent models of renal failure, adenine diet induced and 5/6 nephrectomy. Moreover, FGF23-treated bone cells required Klotho to increase FGF23 mRNA and ERK phosphorylation. In summary, our novel findings show that Klotho in bone is crucial for inducing FGF23 production upon renal failure. We propose the presence of an autocrine feedback loop in which Klotho senses the need for FGF23.-Kaludjerovic, J., Komaba, H., Sato, T., Erben, R. G., Baron, R., Olauson, H., Larsson, T. E., Lanske, B. Klotho expression in long bones regulates FGF23 production during renal failure. © FASEB.

  18. Regulation of cerebrospinal fluid production by caffeine consumption

    Directory of Open Access Journals (Sweden)

    Yoon Sik

    2009-09-01

    Full Text Available Abstract Background Caffeine is the most commonly consumed psycho-stimulant in the world. The effects of caffeine on the body have been extensively studied; however, its effect on the structure of the brain has not been investigated to date. Results In the present study we found that the long-term consumption of caffeine can induce ventriculomegaly; this was observed in 40% of the study rats. In the caffeine-treated rats with ventriculomegaly, there was increased production of CSF, associated with the increased expression of Na+, K+-ATPase and increased cerebral blood flow (CBF. In contrast to the chronic effects, acute treatment with caffeine decreased the production of CSF, suggesting 'effect inversion' associated with caffeine, which was mediated by increased expression of the A1 adenosine receptor, in the choroid plexus of rats chronically treated with caffeine. The involvement of the A1 adenosine receptor in the effect inversion of caffeine was further supported by the induction of ventriculomegaly and Na+, K+-ATPase, in A1 agonist-treated rats. Conclusion The results of this study show that long-term consumption of caffeine can induce ventriculomegaly, which is mediated in part by increased production of CSF. Moreover, we also showed that adenosine receptor signaling can regulate the production of CSF by controlling the expression of Na+, K+-ATPase and CBF.

  19. Metabolic Regulation of Natural Killer Cell IFN-γ Production.

    Science.gov (United States)

    Mah, Annelise Y; Cooper, Megan A

    2016-01-01

    Metabolism is critical for a host of cellular functions and provides a source of intracellular energy. It has been recognized recently that metabolism also regulates differentiation and effector functions of immune cells. Although initial work in this field has focused largely on T lymphocytes, recent studies have demonstrated metabolic control of innate immune cells, including natural killer (NK) cells. Here, we review what is known regarding the metabolic requirements for NK cell activation, focusing on NK cell production of interferon-gamma (IFN-γ). NK cells are innate immune lymphocytes that are poised for rapid activation during the early immune response. Although their basal metabolic rates do not change with short-term activation, they exhibit specific metabolic requirements for activation depending upon the stimulus received. These metabolic requirements for NK cell activation are altered by culturing NK cells with interleukin-15, which increases NK cell metabolic rates at baseline and shifts them toward aerobic glycolysis. We discuss the metabolic pathways important for NK cell production of IFN-γ protein and potential mechanisms whereby metabolism regulates NK cell function.

  20. Transport and transcriptional regulation of oil production in plants.

    Science.gov (United States)

    Manan, Sehrish; Chen, Beibei; She, Guangbiao; Wan, Xiaochun; Zhao, Jian

    2017-08-01

    Triacylglycerol (TAG) serves as an energy reservoir and phospholipids as build blocks of biomembrane to support plant life. They also provide human with foods and nutrients. Multi-compartmentalized biosynthesis, trafficking or cross-membrane transport of lipid intermediates or precursors and their regulatory mechanisms are not fully understood. Recent progress has aided our understanding of how fatty acids (FAs) and phospholipids are transported between the chloroplast, the cytoplasm, and the endoplasmic reticulum (ER), and how the ins and outs of lipids take place in the peroxisome and other organelles for lipid metabolism and function. In addition, information regarding the transcriptional regulation network associated with FA and TAG biosynthesis has been further enriched. Recent breakthroughs made in lipid transport and transcriptional regulation has provided significant insights into our comprehensive understanding of plant lipid biology. This review attempts to highlight the recent progress made on lipid synthesis, transport, degradation, and their regulatory mechanisms. Metabolic engineering, based on these knowledge-powered technologies for production of edible oils or biofuels, is reviewed. The biotechnological application of metabolic enzymes, transcription factors and transporters, for oil production and composition improvement, are discussed in a broad context in order to provide a fresh scenario for researchers and to guide future research and applications.

  1. High brain ammonia tolerance and down-regulation of Na+:K+:2Cl(- Cotransporter 1b mRNA and protein expression in the brain of the Swamp Eel, Monopterus albus, exposed to environmental ammonia or terrestrial conditions.

    Directory of Open Access Journals (Sweden)

    Yuen K Ip

    Full Text Available Na(+:K(+:2Cl(- cotransporter 1 (NKCC1 has been implicated in mediating ischemia-, trauma- or ammonia-induced astrocyte swelling/brain edema in mammals. This study aimed to determine the effects of ammonia or terrestrial exposure on ammonia concentrations in the plasma and brain, and the mRNA expression and protein abundance of nkcc/Nkcc in the brain, of the swamp eel Monopterusalbus. Ammonia exposure led to a greater increase in the ammonia concentration in the brain of M. albus than terrestrial exposure. The brain ammonia concentration of M. albus reached 4.5 µmol g(-1 and 2.7 µmol g(-1 after 6 days of exposure to 50 mmol l(-1 NH4Cl and terrestrial conditions, respectively. The full cDNA coding sequence of nkcc1b from M. albus brain comprised 3276 bp and coded for 1092 amino acids with an estimated molecular mass of 119.6 kDa. A molecular characterization indicated that it could be activated through phosphorylation and/or glycosylation by osmotic and/or oxidative stresses. Ammonia exposure for 1 day or 6 days led to significant decreases in the nkcc1b mRNA expression and Nkcc1b protein abundance in the brain of M. albus. In comparison, a significant decrease in nkcc1b mRNA expression was observed in the brain of M. albus only after 6 days of terrestrial exposure, but both 1 day and 6 days of terrestrial exposure resulted in significant decreases in the protein abundance of Nkcc1b. These results are novel because it has been established in mammals that ammonia up-regulates NKCC1 expression in astrocytes and NKCC1 plays an important role in ammonia-induced astrocyte swelling and brain edema. By contrast, our results indicate for the first time that M. albus is able to down-regulate the mRNA and protein expression of nkcc1b/Nkcc1b in the brain when confronted with ammonia toxicity, which could be one of the contributing factors to its extraordinarily high brain ammonia tolerance.

  2. Regulation of staphylococcal enterotoxin B-elicited nitric oxide production by endothelial cells.

    Science.gov (United States)

    LeClaire, R D; Kell, W M; Sadik, R A; Downs, M B; Parker, G W

    1995-01-01

    The effect of staphylococcal enterotoxin B (SEB)-elicited inducible nitric oxide synthase (iNOS) in mouse endothelial cells was investigated. Results showed that SEB stimulated the same level of NO production in gamma interferon (IFN-gamma)-primed cells as did trichloroacetic acid-extracted lipopolysaccharide. The kinetics of induced NO production and expression of mRNA for iNOS differed markedly in endothelial and macrophage cells. Induced endothelial nitrite production was transient and was 15 to 20% of that generated by macrophage cells; mRNA levels peaked by 2 h and then steadily declined, whereas macrophage message levels continually increased. The ability of endothelial cells to produce SEB-induced NO depended on priming with IFN-gamma, although detectable mRNA could be elicited by SEB alone. Induction of endothelial iNOS mRNA was inhibited by cycloheximide, which indicated a requirement for de novo protein synthesis. Niacinamide and interleukin-10 significantly reduced SEB-induced endothelial NO production. Both are reported to affect IFN-gamma-induced class II major histocompatibility complex (MHC) expression on antigen-presenting cells. Niacinamide reduced iNOS mRNA levels and markedly reduced IFN-gamma induction of endothelial class II MHC surface antigen. Interleukin-10 did not consistently reduce iNOS mRNA expression and had no effect on IFN-gamma induction of endothelial class II MHC surface antigen. These results suggest that SEB interacts with IFN-gamma-primed endothelial cells to elicit induced NO and that this induction can be effectively modulated at the receptor or transcriptional level. PMID:7529748

  3. Temporal regulation of HTLV-2 expression in infected cell lines and patients: evidence for distinct expression kinetics with nuclear accumulation of APH-2 mRNA

    Directory of Open Access Journals (Sweden)

    Bender Cecilia

    2012-09-01

    Full Text Available Abstract Background Human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2 are delta retroviruses with similar genetic organization. Although both viruses immortalize T-cells in vitro, they exhibit distinct pathogenic potential in vivo. To search for possible differences in its expression strategy with respect to HTLV-1, we investigated the pattern of HTLV-2 expression in infected cell lines and peripheral blood mononuclear cells (PBMCs from infected patients using splice site-specific quantitative RT-PCR. Findings A novel alternative splice acceptor site for exon 2 was identified; its usage in env transcripts was found to be subtype-specific. Time-course analysis revealed a two-phase expression kinetics in an infected cell line and in PBMCs of two of the three patients examined; this pattern was reminiscent of HTLV-1. In addition, the minus-strand APH2 transcript was mainly detected in the nucleus, a feature that was similar to its HTLV-1 orthologue HBZ. In contrast to HTLV-1, expression of the mRNA encoding the main regulatory proteins Tax and Rex and that of the mRNAs encoding the p28 and truncated Rex inhibitors is skewed towards p28/truncated Rex inhibitors in HTLV-2. Conclusion Our data suggest a general converging pattern of expression of HTLV-2 and HTLV-1 and highlight peculiar differences in the expression of regulatory proteins that might influence the pathobiology of these viruses.

  4. EU Regulation of Nanobiocides: Challenges in Implementing the Biocidal Product Regulation (BPR)

    DEFF Research Database (Denmark)

    Brinch, Anna; Hansen, Steffen Foss; Hartmann, Nanna B.

    2016-01-01

    The Biocidal Products Regulation (BPR) contains several provisions for nanomaterials (NMs) and is the first regulation in the European Union to require specific testing and risk assessment for the NM form of a biocidal substance as a part of the information requirements. Ecotoxicological data...... are one of the pillars of the information requirements in the BPR, but there are currently no standard test guidelines for the ecotoxicity testing of NMs. The overall objective of this work was to investigate the implications of the introduction of nano-specific testing requirements in the BPR...... and to explore how these might be fulfilled in the case of copper oxide nanoparticles. While there is information and data available in the open literature that could be used to fulfill the BPR information requirements, most of the studies do not take the Organisation for Economic Co-operation and Development...

  5. Expression profile of peripheral tissue antigen genes in medullary thymic epithelial cells (mTECs) is dependent on mRNA levels of autoimmune regulator (Aire).

    Science.gov (United States)

    Oliveira, Ernna H; Macedo, Claudia; Donate, Paula B; Almeida, Renata S; Pezzi, Nicole; Nguyen, Catherine; Rossi, Marcos A; Sakamoto-Hojo, Elza T; Donadi, Eduardo A; Passos, Geraldo A

    2013-01-01

    In the thymus of non-obese diabetic (NOD) mice, the expression of the autoimmune regulator (Aire) gene varies with age, and its down-regulation in young mice precedes the later emergence of type 1 diabetes mellitus (T1D). In addition, the insulin (Ins2) peripheral tissue antigen (PTA) gene, which is Aire-dependent, is also deregulated in these mice. Based in these findings, we hypothesized that the imbalance in PTA gene expression in the thymus can be associated with slight variations in Aire transcript levels. To test this, we used siRNA to knockdown Aire by in vivo electro-transfection of the thymus of BALB/c mice. The efficiency of the electro-transfection was monitored by assessing the presence of irrelevant Cy3-labeled siRNA in the thymic stroma. Importantly, Aire-siRNA reached medullary thymic epithelial cells (mTECs) down-regulating Aire. As expected, the in vivo Aire knockdown was partial and transient; the maximum 59% inhibition occurred in 48 h. The Aire knockdown was sufficient to down-regulate PTA genes; however, surprisingly, several others, including Ins2, were up-regulated. The modulation of these genes after in vivo Aire knockdown was comparable to that observed in NOD mice before the emergence of T1D. The in vitro transfections of 3.10 mTEC cells with Aire siRNA resulted in samples featuring partial (69%) and complete (100%) Aire knockdown. In these Aire siRNA-transfected 3.10 mTECs, the expression of PTA genes, including Ins2, was down-regulated. This suggests that the expression profile of PTA genes in mTECs is affected by fine changes in the transcription level of Aire. Copyright © 2012 Elsevier GmbH. All rights reserved.

  6. Differential feedback regulation of cholesterol 7α-hydroxylase mRNA and transcriptional activity by rat bile acids in primary monolayer cultures of rat hepatocytes

    NARCIS (Netherlands)

    Twisk, J.; Lehmann, E.M.; Princen, H.M.G.

    1993-01-01

    We have used primary monolayer cultures of rat hepatocytes to study the effects of physiological concentrations of various bile acids, commonly found in bile of normal rats, on the mechanism of regulation of cholesterol 7α-hydroxylase and bile acid synthesis. Addition of taurocholic acid, the most

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

    Science.gov (United States)

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

    2015-08-01

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

  8. Postexercise cold water immersion modulates skeletal muscle PGC-1α mRNA expression in immersed and nonimmersed limbs: evidence of systemic regulation.

    Science.gov (United States)

    Allan, Robert; Sharples, Adam P; Close, Graeme L; Drust, Barry; Shepherd, Sam O; Dutton, John; Morton, James P; Gregson, Warren

    2017-08-01

    Mechanisms mediating postexercise cold-induced increases in PGC-1α gene expression in human skeletal muscle are yet to be fully elucidated but may involve local cooling effects on AMPK and p38 MAPK-related signaling and/or increased systemic β-adrenergic stimulation. Therefore, we aimed to examine whether postexercise cold water immersion enhancement of PGC-1α mRNA is mediated through local or systemic mechanisms. Ten subjects completed acute cycling (8 × 5 min at ~80% peak power output) followed by seated-rest (CON) or single-leg cold water immersion (CWI; 10 min, 8°C). Muscle biopsies were obtained preexercise, postexercise, and 3 h postexercise from a single limb in the CON condition but from both limbs in CWI [thereby providing tissue from a CWI and nonimmersed limb (NOT)]. Muscle temperature decreased up to 2 h postexercise following CWI (-5°C) in the immersed limb, with lesser changes observed in CON and NOT (-3°C, P cold induction of PGC-1α mRNA.NEW & NOTEWORTHY We report for the first time that postexercise cold water immersion of one limb also enhances PGC-1α expression in a contralateral, nonimmersed limb. We suggest that increased systemic β-adrenergic stimulation, and not localized cooling per se, exerts regulatory effects on local signaling cascades, thereby modulating PGC-1α expression. Therefore, these data have important implications for research designs that adopt contralateral, nonimmersed limbs as a control condition while also increasing our understanding of the potential mechanisms underpinning cold-mediated PGC-1α responses. Copyright © 2017 the American Physiological Society.

  9. Suppression of Cancer Stemness p21-regulating mRNA and microRNA Signatures in Recurrent Ovarian Cancer Patient Samples

    LENUS (Irish Health Repository)

    Gallagher, Michael F

    2012-01-19

    Abstract Background Malignant ovarian disease is characterised by high rates of mortality due to high rates of recurrent chemoresistant disease. Anecdotal evidence indicates this may be due to chemoresistant properties of cancer stem cells (CSCs). However, our understanding of the role of CSCs in recurrent ovarian disease remains sparse. In this study we used gene microarrays and meta-analysis of our previously published microRNA (miRNA) data to assess the involvement of cancer stemness signatures in recurrent ovarian disease. Methods Microarray analysis was used to characterise early regulation events in an embryonal carcinoma (EC) model of cancer stemness. This was then compared to our previously published microarray data from a study of primary versus recurrent ovarian disease. In parallel, meta-analysis was used to identify cancer stemness miRNA signatures in tumor patient samples. Results Microarray analysis demonstrated a 90% difference between gene expression events involved in early regulation of differentiation in murine EC (mEC) and embryonic stem (mES) cells. This contrasts the known parallels between mEC and mES cells in the undifferentiated and well-differentiated states. Genelist comparisons identified a cancer stemness signature set of genes in primary versus recurrent data, a subset of which are known p53-p21 regulators. This signature is present in primary and recurrent or in primary alone but essentially never in recurrent tumors specifically. Meta-analysis of miRNA expression showed a much stronger cancer stemness signature within tumor samples. This miRNA signature again related to p53-p21 regulation and was expressed prominently in recurrent tumors. Our data indicate that the regulation of p53-p21 in ovarian cancer involves, at least partially, a cancer stemness component. Conclusion We present a p53-p21 cancer stemness signature model for ovarian cancer. We propose that this may, at least partially, differentially regulate the p53-p21

  10. SERCA pump activity is physiologically regulated by presenilin and regulates amyloid beta production.

    Science.gov (United States)

    Green, Kim N; Demuro, Angelo; Akbari, Yama; Hitt, Brian D; Smith, Ian F; Parker, Ian; LaFerla, Frank M

    2008-06-30

    In addition to disrupting the regulated intramembraneous proteolysis of key substrates, mutations in the presenilins also alter calcium homeostasis, but the mechanism linking presenilins and calcium regulation is unresolved. At rest, cytosolic Ca(2+) is maintained at low levels by pumping Ca(2+) into stores in the endoplasmic reticulum (ER) via the sarco ER Ca(2+)-ATPase (SERCA) pumps. We show that SERCA activity is diminished in fibroblasts lacking both PS1 and PS2 genes, despite elevated SERCA2b steady-state levels, and we show that presenilins and SERCA physically interact. Enhancing presenilin levels in Xenopus laevis oocytes accelerates clearance of cytosolic Ca(2+), whereas higher levels of SERCA2b phenocopy PS1 overexpression, accelerating Ca(2+) clearance and exaggerating inositol 1,4,5-trisphosphate-mediated Ca(2+) liberation. The critical role that SERCA2b plays in the pathogenesis of Alzheimer's disease is underscored by our findings that modulating SERCA activity alters amyloid beta production. Our results point to a physiological role for the presenilins in Ca(2+) signaling via regulation of the SERCA pump.

  11. A long non-coding RNA, LncMyoD, regulates skeletal muscle differentiation by blocking IMP2-mediated mRNA translation.

    Science.gov (United States)

    Gong, Chenguang; Li, Zhizhong; Ramanujan, Krishnan; Clay, Ieuan; Zhang, Yunyu; Lemire-Brachat, Sophie; Glass, David J

    2015-07-27

    Increasing evidence suggests that long non-coding RNAs (LncRNAs) represent a new class of regulators of stem cells. However, the roles of LncRNAs in stem cell maintenance and myogenesis remain largely unexamined. For this study, hundreds of intergenic LncRNAs were identified that are expressed in myoblasts and regulated during differentiation. One of these LncRNAs, termed LncMyoD, is encoded next to the Myod gene and is directly activated by MyoD during myoblast differentiation. Knockdown of LncMyoD strongly inhibits terminal muscle differentiation, largely due to a failure to exit the cell cycle. LncMyoD directly binds to IGF2-mRNA-binding protein 2 (IMP2) and negatively regulates IMP2-mediated translation of proliferation genes such as N-Ras and c-Myc. While the RNA sequence of LncMyoD is not well conserved between human and mouse, its locus, gene structure, and function are preserved. The MyoD-LncMyoD-IMP2 pathway elucidates a mechanism as to how MyoD blocks proliferation to create a permissive state for differentiation. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Plant Glutathione Biosynthesis: Diversity in Biochemical Regulation and Reaction Products

    Directory of Open Access Journals (Sweden)

    Ashley eGalant

    2011-09-01

    Full Text Available In plants, exposure to temperature extremes, heavy metal-contaminated soils, drought, air pollutants, and pathogens results in the generation of reactive oxygen species that alter the intracellular redox environment, which in turn influences signaling pathways and cell fate. As part of their response to these stresses, plants produce glutathione. Glutathione acts as an antioxidant by quenching reactive oxygen species, and is involved in the ascorbate-glutathione cycle that eliminates damaging peroxides. Plants also use glutathione for the detoxification of xenobiotics, herbicides, air pollutants (sulfur dioxide and ozone, and toxic heavy metals. Two enzymes catalyze glutathione synthesis: glutamate-cysteine ligase (GCL, and glutathione synthetase (GS. Glutathione is a ubiquitous protective compound in plants, but the structural and functional details of the proteins that synthesize it, as well as the potential biochemical mechanisms of their regulation, have only begun to be explored. As discussed here, the core reactions of glutathione synthesis are conserved across various organisms, but plants have diversified both the regulatory mechanisms that control its synthesis and the range of products derived from this pathway. Understanding the molecular basis of glutathione biosynthesis and its regulation will expand our knowledge of this component in the plant stress response network.

  13. Verification of product design using regulation knowledge base and Web services

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ik June [KAERI, Daejeon (Korea, Republic of); Lee, Jae Chul; Mun Du Hwan [Kyungpook National University, Daegu (Korea, Republic of); Kim, Byung Chul [Dong-A University, Busan (Korea, Republic of); Hwang, Jin Sang [PartDB Co., Ltd., Daejeom (Korea, Republic of); Lim, Chae Ho [Korea Institute of Industrial Technology, Incheon (Korea, Republic of)

    2015-11-15

    Since product regulations contain important rules or codes that manufacturers must follow, automatic verification of product design with the regulations related to a product is necessary. For this, this study presents a new method for the verification of product design using regulation knowledge base and Web services. Regulation knowledge base consisting of product ontology and rules was built with a hybrid technique combining ontology and programming languages. Web service for design verification was developed ensuring the flexible extension of knowledge base. By virtue of two technical features, design verification is served to various products while the change of system architecture is minimized.

  14. Regulation of a plant pathogenesis-related enzyme: Inhibition of chitinase and chitinase mRNA accumulation in cultured tobacco tissues by auxin and cytokinin

    OpenAIRE

    Shinshi, Hideaki; Mohnen, Debra; Meins, Frederick

    1987-01-01

    Two endochitinases (EC 3.2.1.14) of Mr values of ≈34,000 and ≈32,000 have been purified from cultured tissues of Nicotiana tabacum cv. Havana 425. The chitinase content of cloned tobacco pith tissues subcultured on hormone-free medium increases by ≈5-fold to 8% of the soluble protein over a 7-day period. This induction is inhibited >90% by addition of combinations of the plant hormones auxin and cytokinin to the culture medium. Chitinase is also developmentally regulated in the intact plant. ...

  15. An alternatively spliced mRNA from the AP-2 gene encodes a negative regulator of transcriptional activation by AP-2.

    OpenAIRE

    Buettner, R; Kannan, P.; Imhof, A.; Bauer, R.; Yim, S O; Glockshuber, R; Van Dyke, M W; Tainsky, M A

    1993-01-01

    AP-2 is a retinoic acid-inducible and developmentally regulated activator of transcription. We have cloned an alternative AP-2 transcript (AP-2B) from the human teratocarcinoma cell line PA-1, which encodes a protein differing in the C terminus from the previously isolated AP-2 protein (AP-2A). This protein contains the activation domain of AP-2 and part of the DNA binding domain but lacks the dimerization domain which is necessary for DNA binding. Analysis of overlapping genomic clones spann...

  16. Production and transcriptional regulation of proanthocyanidin biosynthesis in forage legumes.

    Science.gov (United States)

    Zhou, Meiliang; Wei, Li; Sun, Zhanmin; Gao, Lihua; Meng, Yu; Tang, Yixiong; Wu, Yanmin

    2015-05-01

    Proanthocyanidins (PA), also known as condensed tannins, contribute to important forage legumes traits including disease resistance and forage quality. PA in forage plants has both positive and negative effects on feed digestibility and animal performance. The analytical methods and their applicability in measuring the contents of PA in forage plants are essential to studies on their nutritional effects. In spite of important breakthroughs in our understanding of the PA biosynthesis, important questions still remain to be answered such as the PA polymerization and transport. Recent advances in the understanding of transcription factor-mediated gene regulation mechanisms in anthocyanin and PA biosynthetic pathway in model plants suggest new approaches for the metabolic engineering of PA in forage plants. The present review will attempt to present the state-of-the-art of research in these areas and provide an update on the production and metabolic engineering of PA in forage plants. We hope that this will contribute to a better understanding of the ways in which PA production to manipulate the content of PA for beneficial effects in forage plants.

  17. Occupational health hazards of trichloroethylene among workers in relation to altered mRNA expression of cell cycle regulating genes (p53, p21, bax and bcl-2) and PPARA.

    Science.gov (United States)

    Varshney, Meenu; Chandra, Abhijit; Jain, Rajeev; Ahmad, Riaz; Bihari, Vipin; Chandran, C Keshava; Mudiam, Mohana K R; Patnaik, Satykam; Goel, S K

    2015-01-01

    Trichloroethylene (TCE) is widely used as a metal degreaser in industrial processes. The present study reports on the effects of TCE exposure on workers employed in the lock industries. To ensure exposure of the workers to TCE, its toxic metabolites, trichloroacetic acid (TCA), dichloroacetic acid (DCA) and trichloroethanol (TCEOH) were detected in the plasma of the subjects through solid phase microextraction-gas chromatography-electron capture detection. TCA, DCA and TCEOH were detected in the range of 0.004-2.494 μg/mL, 0.01-3.612 μg/mL and 0.002-0.617 μg/mL, respectively. Quantitative reverse transcription polymerase chain reaction analysis revealed up-regulated expression of p53 (2.4-fold; p  < 0.05), p21 (2-fold; p  < 0.01), bax (2.9-fold; p  < 0.01) mRNAs and down-regulated expression of bcl-2 (67%; p  < 0.05) mRNAs, indicating DNA damaging potential of these metabolites. No effects were observed on the levels of p16 and c-myc mRNAs. Further, as TCA and DCA, the ligand of peroxisome proliferator activated receptor alpha (PPARA), are involved in the process of hepatocarcinogenesis in rodents, we examined expression of PPARA mRNA and let-7c miRNA in the workers. No statistically significant differences in expression of PPARA mRNA and let-7c miRNA in patients were observed as compared to values in controls. Dehydroepiandosterone sulfate (DHEAS) is a reported endogenous ligand of PPARA so its competitive role was also studied. We observed decreased levels of DHEAS hormone in the subjects. Hence, its involvement in mediation of the observed changes in the levels of various mRNAs analyzed in this study appears unlikely.

  18. The fermentation stress response protein Aaf1p/Yml081Wp regulates acetate production in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Christopher J Walkey

    Full Text Available The production of acetic acid during wine fermentation is a critical issue for wineries since the sensory quality of a wine can be affected by the amount of acetic acid it contains. We found that the C2H2-type zinc-finger transcription factor YML081Wp regulated the mRNA levels of ALD4 and ALD6, which encode a cytosolic acetaldehyde dehydrogenase (ACDH and a mitochondrial ACDH, respectively. These enzymes produce acetate from acetaldehyde as part of the pyruvate dehydrogenase bypass. This regulation was also reflected in the protein levels of Ald4p and Ald6p, as well as total ACDH activity. In the absence of ALD6, YML081W had no effect on acetic acid levels, suggesting that this transcription factor's effects are mediated primarily through this gene. lacZ reporter assays revealed that Yml081wp stimulates ALD6 transcription, in large part from a GAGGGG element 590 base pairs upstream of the translation start site. The non-annotated ORF YML081W therefore encodes a transcription factor that regulates acetate production in Saccharomyces cerevisiae. We propose AAF1 as a gene name for the YML081W ORF.

  19. Regulatory Elements Located in the Upstream Region of the Rhizobium leguminosarum rosR Global Regulator Are Essential for Its Transcription and mRNA Stability

    Directory of Open Access Journals (Sweden)

    Kamila Rachwał

    2017-12-01

    Full Text Available Rhizobium leguminosarum bv. trifolii is a soil bacterium capable of establishing a symbiotic relationship with clover (Trifolium spp.. Previously, the rosR gene, encoding a global regulatory protein involved in motility, synthesis of cell-surface components, and other cellular processes was identified and characterized in this bacterium. This gene possesses a long upstream region that contains several regulatory motifs, including inverted repeats (IRs of different lengths. So far, the role of these motifs in the regulation of rosR transcription has not been elucidated in detail. In this study, we performed a functional analysis of these motifs using a set of transcriptional rosR-lacZ fusions that contain mutations in these regions. The levels of rosR transcription for different mutant variants were evaluated in R. leguminosarum using both quantitative real-time PCR and β-galactosidase activity assays. Moreover, the stability of wild type rosR transcripts and those with mutations in the regulatory motifs was determined using an RNA decay assay and plasmids with mutations in different IRs located in the 5′-untranslated region of the gene. The results show that transcription of rosR undergoes complex regulation, in which several regulatory elements located in the upstream region and some regulatory proteins are engaged. These include an upstream regulatory element, an extension of the -10 element containing three nucleotides TGn (TGn-extended -10 element, several IRs, and PraR repressor related to quorum sensing.

  20. Polysome Fractionation to Analyze mRNA Distribution Profiles.

    Science.gov (United States)

    Panda, Amaresh C; Martindale, Jennifer L; Gorospe, Myriam

    2017-02-05

    Eukaryotic cells adapt to changes in external or internal signals by precisely modulating the expression of specific gene products. The expression of protein-coding genes is controlled at the transcriptional and post-transcriptional levels. Among the latter steps, the regulation of translation is particularly important in cellular processes that require rapid changes in protein expression patterns. The translational efficiency of mRNAs is altered by RNA-binding proteins (RBPs) and noncoding (nc)RNAs such as microRNAs (Panda et al., 2014a and 2014b; Abdelmohsen et al., 2014). The impact of factors that regulate selective mRNA translation is a critical question in RNA biology. Polyribosome (polysome) fractionation analysis is a powerful method to assess the association of ribosomes with a given mRNA. It provides valuable information about the translational status of that mRNA, depending on the number of ribosomes with which they are associated, and identifies mRNAs that are not translated (Panda et al., 2016). mRNAs associated with many ribosomes form large polysomes that are predicted to be actively translated, while mRNAs associated with few or no ribosomes are expected to be translated poorly if at all. In sum, polysome fractionation analysis allows the direct determination of translation efficiencies at the level of the whole transcriptome as well as individual mRNAs.

  1. Computational Analysis of mRNA Expression Profiles Identifies the ITG Family and PIK3R3 as Crucial Genes for Regulating Triple Negative Breast Cancer Cell Migration

    Directory of Open Access Journals (Sweden)

    Sukhontip Klahan

    2014-01-01

    Full Text Available Triple-negative breast cancer (TNBC is an aggressive type of breast cancer that does not express estrogen receptor (ER, progesterone receptor (PR, and human epidermal growth factor receptor (Her2/neu. TNBC has worse clinical outcomes than other breast cancer subtypes. However, the key molecules and mechanisms of TNBC migration remain unclear. In this study, we compared two normalized microarray datasets from GEO database between Asian (GSE33926 and non-Asian populations (GSE46581 to determine the molecules and common pathways in TNBC migration. We demonstrated that 16 genes in non-Asian samples and 9 genes in Asian samples are related to TNBC migration. In addition, our analytic results showed that 4 genes, PIK3R3, ITGB1, ITGAL, and ITGA6, were involved in the regulation of actin cytoskeleton. Our results indicated potential genes that link to TNBC migration. This study may help identify novel therapeutic targets for drug development in cancer therapy.

  2. Cortisol regulation of ion transporter mRNA in Atlantic salmon gill and the effect of salinity on the signaling pathway

    DEFF Research Database (Denmark)

    Kiilerich, Pia; Kristiansen, Karsten; Madsen, Steffen S

    2007-01-01

    in gills from freshwater (FW) and seawater (SW) acclimated fish. The effect of cortisol was highly dependent on acclimation to salinity. In FW, cortisol stimulated the transcript levels of CFTR-I and Na(+), K(+)-ATPase alpha1a and alpha1b. Addition of RU486 totally abolished the cortisol effects on CFTR...... and the associated receptor signaling pathway (glucocorticoid (GR) versus mineralocorticoid (MR)) of cortisol was investigated in the salmon gill. Using a gill organ culture technique, the effect of cortisol with and without addition of specific hormone receptor antagonists (RU486 and spironolactone) was analyzed......-I and Na(+), K(+)-ATPase alpha1b, suggesting that signaling was mediated via GR. Interestingly, both spironolactone and RU486 were able to inhibit the cortisol effect on Na(+), K(+)-ATPase alpha1a indicating a role for both MR and GR in regulation of this target gene. In SW, cortisol increased...

  3. Evolution of RLSB, a nuclear-encoded S1 domain RNA binding protein associated with post-transcriptional regulation of plastid-encoded rbcL mRNA in vascular plants.

    Science.gov (United States)

    Yerramsetty, Pradeep; Stata, Matt; Siford, Rebecca; Sage, Tammy L; Sage, Rowan F; Wong, Gane Ka-Shu; Albert, Victor A; Berry, James O

    2016-06-29

    RLSB, an S-1 domain RNA binding protein of Arabidopsis, selectively binds rbcL mRNA and co-localizes with Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) within chloroplasts of C3 and C4 plants. Previous studies using both Arabidopsis (C3) and maize (C4) suggest RLSB homologs are post-transcriptional regulators of plastid-encoded rbcL mRNA. While RLSB accumulates in all Arabidopsis leaf chlorenchyma cells, in C4 leaves RLSB-like proteins accumulate only within Rubisco-containing bundle sheath chloroplasts of Kranz-type species, and only within central compartment chloroplasts in the single cell C4 plant Bienertia. Our recent evidence implicates this mRNA binding protein as a primary determinant of rbcL expression, cellular localization/compartmentalization, and photosynthetic function in all multicellular green plants. This study addresses the hypothesis that RLSB is a highly conserved Rubisco regulatory factor that occurs in the chloroplasts all higher plants. Phylogenetic analysis has identified RLSB orthologs and paralogs in all major plant groups, from ancient liverworts to recent angiosperms. RLSB homologs were also identified in algae of the division Charophyta, a lineage closely related to land plants. RLSB-like sequences were not identified in any other algae, suggesting that it may be specific to the evolutionary line leading to land plants. The RLSB family occurs in single copy across most angiosperms, although a few species with two copies were identified, seemingly randomly distributed throughout the various taxa, although perhaps correlating in some cases with known ancient whole genome duplications. Monocots of the order Poales (Poaceae and Cyperaceae) were found to contain two copies, designated here as RLSB-a and RLSB-b, with only RLSB-a implicated in the regulation of rbcL across the maize developmental gradient. Analysis of microsynteny in angiosperms revealed high levels of conservation across eudicot species and for both paralogs in

  4. cAMP/PKA regulates osteogenesis, adipogenesis and ratio of RANKL/OPG mRNA expression in mesenchymal stem cells by suppressing leptin.

    Directory of Open Access Journals (Sweden)

    Der-Chih Yang

    Full Text Available BACKGROUND: Mesenchymal stem cells (MSCs are a pluripotent cell type that can differentiate into adipocytes, osteoblasts and other cells. The reciprocal relationship between adipogenesis and osteogenesis was previously demonstrated; however, the mechanisms remain largely unknown. METHODS AND FINDINGS: We report that activation of PKA by 3-isobutyl-1 methyl xanthine (IBMX and forskolin enhances adipogenesis, the gene expression of PPARgamma2 and LPL, and downregulates the gene expression of Runx2 and osteopontin, markers of osteogenesis. PKA activation also decreases the ratio of Receptor Activator of the NF-kappaB Ligand to Osteoprotegerin (RANKL/OPG gene expression - the key factors of osteoclastogenesis. All these effects are mediated by the cAMP/PKA/CREB pathway by suppressing leptin, and may contribute to PKA stimulators-induced in vivo bone loss in developing zebrafish. CONCLUSIONS: Using MSCs, the center of a newly proposed bone metabolic unit, we identified cAMP/PKA signaling, one of the many signaling pathways that regulate bone homeostasis via controlling cyto-differentiation of MSCs and altering RANKL/OPG gene expression.

  5. Mitotic protein kinase CDK1 phosphorylation of mRNA translation regulator 4E-BP1 Ser83 may contribute to cell transformation

    Energy Technology Data Exchange (ETDEWEB)

    Velasquez, Celestino; Cheng, Erdong; Shuda, Masahiro; Lee-Oesterreich, Paula J.; Pogge von Strandmann, Lisa; Gritsenko, Marina A.; Jacobs, Jon M.; Moore, Patrick S.; Chang, Yuan

    2016-07-11

    mTOR-directed 4E-BP1 phosphorylation promotes cap-dependent translation and tumorigen-esis. During mitosis, CDK1 substitutes for mTOR and fully phosphorylates 4E-BP1 at canoni-cal as well a non-canonical S83 site resulting in a mitosis-specific hyperphosphorylated δ isoform. Colocalization studies with a phospho-S83 specific antibody indicate that 4E-BP1 S83 phosphorylation accumulates at centrosomes during prophase, peaks at metaphase, and decreases through telophase. While S83 phosphorylation of 4E-BP1 does not affect in vitro cap-dependent translation, nor eIF4G/4E-BP1 cap-binding, expression of an alanine substitution mutant 4E-BP1.S83A partially reverses rodent cell transformation induced by Merkel cell polyomavirus (MCV) small T (sT) antigen viral oncoprotein. In contrast to inhibitory mTOR 4E-BP1 phosphorylation, these findings suggest that mitotic CDK1-directed phosphorylation of δ-4E-BP1 may yield a gain-of-function, distinct from translation regulation, that may be important in tumorigenesis and mitotic centrosome function.

  6. A selective splicing variant of hepcidin mRNA in hepatocellular carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Toki, Yasumichi [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Sasaki, Katsunori, E-mail: k-sasaki@asahikawa-med.ac.jp [Department of Gastrointestinal Immunology and Regenerative Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Tanaka, Hiroki [Department of Legal Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Yamamoto, Masayo; Hatayama, Mayumi; Ito, Satoshi; Ikuta, Katsuya; Shindo, Motohiro; Hasebe, Takumu; Nakajima, Shunsuke; Sawada, Koji; Fujiya, Mikihiro [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Torimoto, Yoshihiro [Oncology Center, Asahikawa Medical University Hospital, Hokkaido 078-8510 (Japan); Ohtake, Takaaki; Kohgo, Yutaka [Department of Gastroenterology, International University of Health and Welfare Hospital, Tochigi 329-2763 (Japan)

    2016-08-05

    Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. By RT-PCR/agarose gel electrophoresis of hepcidin mRNA in a hepatocellular carcinoma cell line HLF, a smaller mRNA band was shown in addition to the wild-type hepcidin mRNA. From sequencing analysis, this additional band was a selective splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene, producing the transcript that encodes truncated peptide lacking 20 amino acids at the middle of preprohepcidin. In the present study, we used the digital PCR, because such a small amount of variant mRNA was difficult to quantitate by the conventional RT-PCR amplification. Among seven hepatoma-derived cell lines, six cell lines have significant copy numbers of this variant mRNA, but not in one cell line. In the transient transfection analysis of variant-type hepcidin cDNA, truncated preprohepcidin has a different character comparing with native preprohepcidin: its product is insensitive to digestion, and secreted into the medium as a whole preprohepcidin form without maturation. Loss or reduction of function of HAMP gene by aberrantly splicing may be a suitable phenomenon to obtain the proliferating advantage of hepatoma cells. - Highlights: • An aberrant splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene. • Absolute quantification of hepcidin mRNA by digital PCR amplification. • Hepatoma-derived cell lines have significant copies of variant-type hepcidin mRNA. • Truncated preprohepcidin is secreted from cells without posttranslational cleavage.

  7. TIM-1 signaling in B cells regulates antibody production.

    Science.gov (United States)

    Ma, Juan; Usui, Yoshihiko; Takeda, Kazuyoshi; Harada, Norihiro; Yagita, Hideo; Okumura, Ko; Akiba, Hisaya

    2011-03-11

    Members of the T cell Ig and mucin (TIM) family have recently been implicated in the control of T cell-mediated immune responses. In this study, we found TIM-1 expression on anti-IgM- or anti-CD40-stimulated splenic B cells, which was further up-regulated by the combination of anti-IgM and anti-CD40 Abs. On the other hand, TIM-1 ligand was constitutively expressed on B cells and inducible on anti-CD3(+) anti-CD28-stimulated CD4(+) T cells. In vitro stimulation of activated B cells by anti-TIM-1 mAb enhanced proliferation and expression of a plasma cell marker syndecan-1 (CD138). We further examined the effect of TIM-1 signaling on antibody production in vitro and in vivo. Higher levels of IgG2b and IgG3 secretion were detected in the culture supernatants of the anti-TIM-1-stimulated B cells as compared with the control IgG-stimulated B cells. When immunized with T-independent antigen TNP-Ficoll, TNP-specific IgG1, IgG2b, and IgG3 Abs were slightly increased in the anti-TIM-1-treated mice. When immunized with T-dependent antigen OVA, serum levels of OVA-specific IgG2b, IgG3, and IgE Abs were significantly increased in the anti-TIM-1-treated mice as compared with the control IgG-treated mice. These results suggest that TIM-1 signaling in B cells augments antibody production by enhancing B cell proliferation and differentiation. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. Means of regulating combustible materials and products in external walls

    Directory of Open Access Journals (Sweden)

    Mikkola Esko

    2016-01-01

    Full Text Available This report presents proposals for defining means of regulating the use of combustible materials and products in external walls. Required protections are based on the quantities of fire loads and their contribution to fire development. The study is based on life safety and protection of property priorities taking into account reaction to fire classes related to different types of fire loads and fire compartmentation requirements of the adjacent spaces of concern. The proposals include the following main principles in relation to fire-separation requirements: In case of internal fire exposure the protective structure for combustible building parts needs to meet at least half of the fire-separating requirement for the compartment of concern. In case of external fire exposure the protection time requirement can be 15 minutes less than for the internal protection. The proposals are applicable for residential buildings and offices. In case of buildings with longer evacuation times more stringent requirement levels may be considered. For verification of protection performance of fire loads it is proposed to use existing standardized test methods (fire protection ability (K classes and fire-separating function (EI classes validated methods of calculation and/or large scale fire testing.

  9. Exercise intensity-dependent regulation of peroxisome proliferator-activated receptor γ coactivator-1α mRNA abundance is associated with differential activation of upstream signalling kinases in human skeletal muscle

    Science.gov (United States)

    Egan, Brendan; Carson, Brian P; Garcia-Roves, Pablo M; Chibalin, Alexander V; Sarsfield, Fiona M; Barron, Niall; McCaffrey, Noel; Moyna, Niall M; Zierath, Juleen R; O’Gorman, Donal J

    2010-01-01

    Skeletal muscle contraction increases intracellular ATP turnover, calcium flux, and mechanical stress, initiating signal transduction pathways that modulate peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α)-dependent transcriptional programmes. The purpose of this study was to determine if the intensity of exercise regulates PGC-1α expression in human skeletal muscle, coincident with activation of signalling cascades known to regulate PGC-1α transcription. Eight sedentary males expended 400 kcal (1674 kj) during a single bout of cycle ergometer exercise on two separate occasions at either 40% (LO) or 80% (HI) of. Skeletal muscle biopsies from the m. vastus lateralis were taken at rest and at +0, +3 and +19 h after exercise. Energy expenditure during exercise was similar between trials, but the high intensity bout was shorter in duration (LO, 69.9 ± 4.0 min; HI, 36.0 ± 2.2 min, P < 0.05) and had a higher rate of glycogen utilization (P < 0.05). PGC-1α mRNA abundance increased in an intensity-dependent manner +3 h after exercise (LO, 3.8-fold; HI, 10.2-fold, P < 0.05). AMP-activated protein kinase (AMPK) (2.8-fold, P < 0.05) and calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylation (84%, P < 0.05) increased immediately after HI but not LO. p38 mitogen-activated protein kinase (MAPK) phosphorylation increased after both trials (∼2.0-fold, P < 0.05), but phosphorylation of the downstream transcription factor, activating transcription factor-2 (ATF-2), increased only after HI (2.4-fold, P < 0.05). Cyclic-AMP response element binding protein (CREB) phosphorylation was elevated at +3 h after both trials (∼80%, P < 0.05) and class IIa histone deacetylase (HDAC) phosphorylation increased only after HI (2.0-fold, P < 0.05). In conclusion, exercise intensity regulates PGC-1α mRNA abundance in human skeletal muscle in response to a single bout of exercise. This effect is mediated by differential activation of multiple

  10. Exercise intensity-dependent regulation of peroxisome proliferator-activated receptor coactivator-1 mRNA abundance is associated with differential activation of upstream signalling kinases in human skeletal muscle.

    Science.gov (United States)

    Egan, Brendan; Carson, Brian P; Garcia-Roves, Pablo M; Chibalin, Alexander V; Sarsfield, Fiona M; Barron, Niall; McCaffrey, Noel; Moyna, Niall M; Zierath, Juleen R; O'Gorman, Donal J

    2010-05-15

    Skeletal muscle contraction increases intracellular ATP turnover, calcium flux, and mechanical stress, initiating signal transduction pathways that modulate peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha)-dependent transcriptional programmes. The purpose of this study was to determine if the intensity of exercise regulates PGC-1alpha expression in human skeletal muscle, coincident with activation of signalling cascades known to regulate PGC-1alpha transcription. Eight sedentary males expended 400 kcal (1674 kj) during a single bout of cycle ergometer exercise on two separate occasions at either 40% (LO) or 80% (HI) of . Skeletal muscle biopsies from the m. vastus lateralis were taken at rest and at +0, +3 and +19 h after exercise. Energy expenditure during exercise was similar between trials, but the high intensity bout was shorter in duration (LO, 69.9 +/- 4.0 min; HI, 36.0 +/- 2.2 min, P < 0.05) and had a higher rate of glycogen utilization (P < 0.05). PGC-1alpha mRNA abundance increased in an intensity-dependent manner +3 h after exercise (LO, 3.8-fold; HI, 10.2-fold, P < 0.05). AMP-activated protein kinase (AMPK) (2.8-fold, P < 0.05) and calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylation (84%, P < 0.05) increased immediately after HI but not LO. p38 mitogen-activated protein kinase (MAPK) phosphorylation increased after both trials (2.0-fold, P < 0.05), but phosphorylation of the downstream transcription factor, activating transcription factor-2 (ATF-2), increased only after HI (2.4-fold, P < 0.05). Cyclic-AMP response element binding protein (CREB) phosphorylation was elevated at +3 h after both trials (80%, P < 0.05) and class IIa histone deacetylase (HDAC) phosphorylation increased only after HI (2.0-fold, P < 0.05). In conclusion, exercise intensity regulates PGC-1alpha mRNA abundance in human skeletal muscle in response to a single bout of exercise. This effect is mediated by differential activation of

  11. Light inhibits gravity-regulated peg formation and asymmetric mRNA accumulation of auxin-inducible CsIAA1 in the cortex of the transition zone in cucumber seedlings

    Science.gov (United States)

    Fujii, Nobuharu; Saito, Yuko; Miyazawa, Yutaka; Takahashi, Hideyuki

    When cucumber seedlings are grown horizontally, a specialized protuberance, termed the peg, develops on the lower side of the transition zone between the hypocotyl and the root. Gravimorphogenesis regulates the lateral positioning of the peg in the transition zone and it has been suggested that auxin plays an important role in peg formation in cucumber seedlings. Here, we found that light inhibited auxin-regulated peg formation. In the transition zone of horizontally positioned cucumber seedlings grown in the dark, we detected an asymmetric accumulation of mRNA from the auxin-inducible gene CsIAA1 in the epidermis and cortex. However, in seedlings grown under illumination, this asymmetry was greatly reduced. In dark- and light-grown seedlings, application of 10 -3 M indole-3-acetic acid induced peg formation on both the lower and upper sides of the transition zone. These results suggest that light inhibits peg formation via modification of auxin distribution and/or levels in the transition zone of cucumber seedlings.

  12. Xplore mRNA assays for the quantification of IL-1 beta and TNF-alpha mRNA in lipopolysaccharide-induced mouse macrophages

    National Research Council Canada - National Science Library

    Van Arsdell, S W; Murphy, K P; Pazmany, C; Erickson, D; Burns, C; Moody, M D

    2000-01-01

    Because the accurate measurement of a number of cytokine mRNA transcripts provides valuable knowledge about cytokine gene regulation, we have developed the Xplore assay for the quantification of cytokine mRNA...

  13. Polymorphism 10034C>T is located in a region regulating polyadenylation of FGG transcripts and influences the fibrinogen gamma'/gammaA mRNA ratio.

    Science.gov (United States)

    Uitte de Willige, S; Rietveld, I M; De Visser, M C H; Vos, H L; Bertina, R M

    2007-06-01

    Fibrinogen gamma haplotype 2 (FGG-H2) is associated with reduced fibrinogen gamma' levels and fibrinogen gamma'/total fibrinogen ratios and with an increased deep-venous thrombosis (DVT) risk. Two FGG-H2 tagging single nucleotide polymorphisms (SNPs), 9615C>T and 10034C>T, are located in the region of alternative FGG pre-mRNA processing. 10034C>T is located in a GT-rich downstream sequence element (DSE) that comprises a putative cleavage stimulation factor (CstF) binding site. To investigate the functionality of SNPs 9615C>T and 10034C>T, and the importance of the DSE containing 10034C>T. Different minigene constructs containing FGG exon 9, intron 9, exon 10 and the 3' region were transiently transfected into HepG2 cells and quantitative real-time polymerase chain reaction was used to measure relative polyadenylation (pA) signal usage (pA1/pA2 ratio). Compared with the reference construct CC (9615C-10034C; FGG-H1; pA1/pA2 ratio set at 100%), the pA1/pA2 ratio of construct TT (9615T-10034T; FGG-H2) was 1.4-fold decreased (71.5%, P = 0.015). The pA1/pA2 ratio of construct CT (9615C-10034T) was almost 1.2-fold decreased (85.3%, P = 0.001), whereas the pA1/pA2 ratio of construct TC (9615T-10034C) did not differ significantly from the reference construct (101.6%, P = 0.890). Functionality of the putative CstF binding site was confirmed using constructs in which this site was deleted or its sequence altered by point mutations. SNP 10034C>T is located in a GT-rich DSE involved in regulating the usage of the pA2 signal of FGG, which may represent a CstF binding site. We propose that the 10034C>T change is the functional variation in FGG-H2 that is responsible for the reduction in the fibrinogen gamma'/total fibrinogen ratio and the increased DVT risk.

  14. Regulation of Apoptosis by Alternative Pre-mRNA Splicing

    National Research Council Canada - National Science Library

    Schwerk, Christian; Schulze-Osthoff, Klaus

    2005-01-01

    .... A large number of apoptotic factors are regulated via alternative splicing, a process that allows for the production of discrete protein isoforms with often distinct functions from a common mRNA precursor...

  15. Administration of JTE013 abrogates experimental asthma by regulating proinflammatory cytokine production from bronchial epithelial cells.

    Science.gov (United States)

    Terashita, Tomomi; Kobayashi, Kazuyuki; Nagano, Tatsuya; Kawa, Yoshitaka; Tamura, Daisuke; Nakata, Kyosuke; Yamamoto, Masatsugu; Tachihara, Motoko; Kamiryo, Hiroshi; Nishimura, Yoshihiro

    2016-11-09

    Sphingosine-1-phosphate (S1P) is a bioactive phospholipid that acts as a signal transducer by binding to S1P receptors (S1PR) 1 to 5. The S1P/S1PRs pathway has been associated with remodeling and allergic inflammation in asthma, but the expression pattern of S1PR and its effects on non-immune cells have not been completely clarified. The aim of this study was to examine the contribution of the signaling of S1P and S1PRs expressed in airway epithelial cells (ECs) to asthma responses in mice. Bronchial asthma was experimentally induced in BALB/c mice by ovalbumin (OVA) sensitization followed by an OVA inhalation challenge. The effects of S1PR antagonists on the development of asthma were analyzed 24 h after the OVA challenge. Immunohistological analysis revealed S1PR1-3 expression on mouse airway ECs. Quantitative real-time polymerase chain reaction demonstrated that S1P greatly stimulated the induction of CCL3 and TIMP2 mRNA in human airway ECs, i.e., BEAS-2B cells, in a dose-dependent manner. Pretreatment with the S1PR2 antagonist JTE013 inhibited the CCL3 gene expression in BEAS-2B cells. Immunohistological analysis also showed that the expression level of CCL3 was attenuated by JTE013 in asthmatic mice. Furthermore, JTE013 as well as anti-CCL3 antibody attenuated allergic responses. Intratracheal administration of JTE013 also attenuated eosinophilic reactions in bronchoalveolar lavage fluids. S1P induced transcription factor NFκB activation, while JTE013 greatly reduced the NFκB activation. JTE013 attenuated allergic airway reactions by regulating CCL3 production from bronchial ECs. The intratracheal administration of JTE013 may be a promising therapeutic strategy for bronchial asthma.

  16. Occupational health hazards of trichloroethylene among workers in relation to altered mRNA expression of cell cycle regulating genes (p53, p21, bax and bcl-2 and PPARA

    Directory of Open Access Journals (Sweden)

    Meenu Varshney

    2015-01-01

    Full Text Available Trichloroethylene (TCE is widely used as a metal degreaser in industrial processes. The present study reports on the effects of TCE exposure on workers employed in the lock industries. To ensure exposure of the workers to TCE, its toxic metabolites, trichloroacetic acid (TCA, dichloroacetic acid (DCA and trichloroethanol (TCEOH were detected in the plasma of the subjects through solid phase microextraction-gas chromatography-electron capture detection. TCA, DCA and TCEOH were detected in the range of 0.004–2.494 μg/mL, 0.01–3.612 μg/mL and 0.002–0.617 μg/mL, respectively. Quantitative reverse transcription polymerase chain reaction analysis revealed up-regulated expression of p53 (2.4-fold; p < 0.05, p21 (2-fold; p < 0.01, bax (2.9-fold; p < 0.01 mRNAs and down-regulated expression of bcl-2 (67%; p < 0.05 mRNAs, indicating DNA damaging potential of these metabolites. No effects were observed on the levels of p16 and c-myc mRNAs. Further, as TCA and DCA, the ligand of peroxisome proliferator activated receptor alpha (PPARA, are involved in the process of hepatocarcinogenesis in rodents, we examined expression of PPARA mRNA and let-7c miRNA in the workers. No statistically significant differences in expression of PPARA mRNA and let-7c miRNA in patients were observed as compared to values in controls. Dehydroepiandosterone sulfate (DHEAS is a reported endogenous ligand of PPARA so its competitive role was also studied. We observed decreased levels of DHEAS hormone in the subjects. Hence, its involvement in mediation of the observed changes in the levels of various mRNAs analyzed in this study appears unlikely.

  17. DIRECTIONS AND MECHANISMS OF STATE REGULATION OF THE PRODUCTION OF FUNCTIONAL BAKERY PRODUCTS

    Directory of Open Access Journals (Sweden)

    I. P. Bogomolova

    2014-01-01

    Full Text Available Summary. In the article the basic socio-economic problems associated with micronutrient deficiency in the diet of the population and their possible solutions through production of a functional purpose, summed up the experience of foreign countries, as well as the possible mechanisms of state regulation and support enterprises of the baking industry in socially significant programs. The rapid pace of development of the economies of most countries of the world have led to negative social consequences, the main one of which is a violation of the power structure in the general population, lack of nutritional vitamins and trace elements , jetlag and diet , eating dangerous and harmful products . Way out of this situation is the creation and distribution of food functionality, including the baking of products. Currently, the production of functional food - one of the fastest growing areas of activity of subjects of the food industry. In enterprises of our region there is a great potential for the development of this segment. By the State in support of this direction are implemented targeted programs aimed at the development of the baking industry for the production of enriched bakery products. The paper presents the principles that can contribute to the effective interaction with state actors bakery business needed to overcome the lag in the development of socially significant sectors of the Russian economy from foreign counterparts, such as the development of competition and the creation of favorable conditions for the realization of entrepreneurial initiatives; increasing the investment attractiveness, social and economic efficiency of production and sale of bread and bakery in the Delhi - regions; reducing administrative barriers to accelerate the development of the market of bread and bakery products functional and specialized purpose . Bakery products needs competent positioning, because the development of the market and changing consumer

  18. Production of pigments in Alternanthera sessilis calli mediated by plant growth regulators and light

    National Research Council Canada - National Science Library

    Cristini Milech; Simone Ribeiro Lucho; Alítcia Moraes Kleinowski; Débora Barwaldt Dutra; Mariana Mühlenberg Soares; Eugenia Jacira Bolacel Braga

    2017-01-01

    .... The objective was to establish callus production in A. sessilis grown under different combinations of growth regulators and light qualities and to assess whether these factors can increase betalain and flavonoid production...

  19. FGF23-regulated production of Fetuin-A (AHSG) in osteocytes.

    Science.gov (United States)

    Mattinzoli, D; Rastaldi, M P; Ikehata, M; Armelloni, S; Pignatari, C; Giardino, L A; Li, M; Alfieri, C M; Regalia, A; Riccardi, D; Messa, P

    2016-02-01

    AHSG, a serum glycoprotein with recognized anti-calcification activity, has also been suggested to modulate both bone formation and resorption. Though the bulk of AHSG is mostly synthesized in the liver, it has been claimed that also bone cells might produce it. However, the extent of the bone AHSG production and the potential controlling factors remain to be definitively proven. A relevant number of studies support the notion that FGF23, a bone-derived hormone, not only regulates the most important mineral metabolism (MM) related factors (phosphate, parathyroid hormone, vitamin D, etc.), but might be also involved in cardiovascular (CV) outcome, both in chronic kidney disease (CKD) patients and in the general population. Furthermore, in addition to some direct autocrine and paracrine effects in bone, FGF23 has been suggested to interact with AHSG. In this study we investigated if AHSG is really produced by bone cells, and if its bone production is related and/or controlled by FGF23, using cultured bone cells, according to a new method recently published by our group. Our data show that AHSG is consistently produced in osteocytes and to a far lesser extent in osteoblasts. Both FGF23 addition to the culture medium and its over-expression in osteocytes were associated with a consistent increase of both AHSG mRNA and protein, while FGF23 silencing was followed by opposite effects. Though most of these results were largely affected by the blockage of FGF23 receptors, the role of these receptors in the different experimental sets is still not completely clarified. In addition, we found that FGF23 and AHSG proteins co-localized both in cytoplasm and nucleus, which suggests a possible reciprocal interactivity. Our data not only confirm that AHSG is produced in bone, mainly in osteocytes, but show for the first time that its production is modulated by FGF23. Since both proteins play important roles in the bone and cardiovascular pathology, these results add new pieces to

  20. Integrin beta 4 MRNA expression levels in bronchial asthma patients ...

    African Journals Online (AJOL)

    Serum total IgE was measured by ELISA and mRNA expression of ITGβ4 was assessed by reverse transcriptase PCR (RT-PCR) using real time PCR.. ITGβ4 mRNA expression was significantly down regulated with increased serum total IgE in patients with asthma compared to controls. Moreover, ITGβ4 expression was ...

  1. Biphasic Modulation of NOS Expression, Protein and Nitrite Products by Hydroxocobalamin Underlies Its Protective Effect in Endotoxemic Shock: Downstream Regulation of COX-2, IL-1β, TNF-α, IL-6, and HMGB1 Expression

    Science.gov (United States)

    Sampaio, André L. F.; Dalli, Jesmond; Brancaleone, Vincenzo; D'Acquisto, Fulvio; Perretti, Mauro; Wheatley, Carmen

    2013-01-01

    Background. NOS/•NO inhibitors are potential therapeutics for sepsis, yet they increase clinical mortality. However, there has been no in vivo investigation of the (in vitro) •NO scavenger, cobalamin's (Cbl) endogenous effects on NOS/•NO/inflammatory mediators during the immune response to sepsis. Methods. We used quantitative polymerase chain reaction (qPCR), ELISA, Western blot, and NOS Griess assays, in a C57BL/6 mouse, acute endotoxaemia model. Results. During the immune response, pro-inflammatory phase, parenteral hydroxocobalamin (HOCbl) treatment partially inhibits hepatic, but not lung, iNOS mRNA and promotes lung eNOS mRNA, but attenuates the LPS hepatic rise in eNOS mRNA, whilst paradoxically promoting high iNOS/eNOS protein translation, but relatively moderate •NO production. HOCbl/NOS/•NO regulation is reciprocally associated with lower 4 h expression of TNF-α, IL-1β, COX-2, and lower circulating TNF-α, but not IL-6. In resolution, 24 h after LPS, HOCbl completely abrogates a major late mediator of sepsis mortality, high mobility group box 1 (HMGB1) mRNA, inhibits iNOS mRNA, and attenuates LPS-induced hepatic inhibition of eNOS mRNA, whilst showing increased, but still moderate, NOS activity, relative to LPS only. experiments (LPS+D-Galactosamine) HOCbl afforded significant, dose-dependent protection in mice Conclusions. HOCbl produces a complex, time- and organ-dependent, selective regulation of NOS/•NO during endotoxaemia, corollary regulation of downstream inflammatory mediators, and increased survival. This merits clinical evaluation. PMID:23781123

  2. 10 CFR 431.402 - Preemption of State regulations for commercial HVAC & WH products.

    Science.gov (United States)

    2010-01-01

    ... 10 Energy 3 2010-01-01 2010-01-01 false Preemption of State regulations for commercial HVAC & WH... regulations for commercial HVAC & WH products. Beginning on the effective date of such standard, an energy conservation standard set forth in this Part for a commercial HVAC & WH product supersedes any State or local...

  3. Expression regulation of design process gene in product design

    DEFF Research Database (Denmark)

    Li, Bo; Fang, Lusheng; Li, Bo

    2011-01-01

    is proposed and analyzed, as well as its three categories i.e., the operator gene, the structural gene and the regulator gene. Second, the trigger mechanism that design objectives and constraints trigger the operator gene is constructed. Third, the expression principle of structural gene is analyzed...... with the example of design management gene. Last, the regulation mode that the regulator gene regulates the expression of the structural gene is established and it is illustrated by taking the design process management gene as an example. © (2011) Trans Tech Publications.......To improve the design process efficiency, this paper proposes the principle and methodology that design process gene controls the characteristics of design process under the framework of design process reuse and optimization based on design process gene. First, the concept of design process gene...

  4. Nerve Growth Factor, Brain-derived Neurotrophic Factor and Osteocalcin gene relationship in energy regulation, bone homeostasis and reproductive organs analyzed by mRNA quantitative evaluation and linear correlation analysis

    Directory of Open Access Journals (Sweden)

    Claudia Camerino

    2016-10-01

    Full Text Available Nerve Growth Factor (NGF / Brain-derived Neurotrophic Factor (BDNF and osteocalcin share common effects regulating energy, bone mass, reproduction and neuronal functions. To investigate on the gene-relationship between NGF, BDNF and Osteocalcin we compared by RT-PCR the transcript levels of Ngf, Bdnf and Osteocalcin as well as of their receptors p75NTR/NTRK1, NTRK2 and Gprc6a in brain, bone, white/brown adipose tissue (WAT/BAT and reproductive organs of 3 months old female and male mice. Brain and bone were used as positive controls for NGF/BDNF and Osteocalcin respectively. The role of oxitocin(Oxt and its receptor(Oxtr was also investigated. Ngf expression shows an opposite trend compared to Bdnf. Ngf/p75NTR expression is 50% higher in BAT than brain, in both genders, but lower in bone. In contrast, Bdnf expression in bone is higher than in brain, but low in BAT/WAT. We found Osteocalcin gene expressed in brain in both genders, but Gprc6a expression is low in brain and BAT/WAT. As expected, Gprc6a gene is expressed in bone. Oxt gene was markedly expressed in brain, Oxtr in the ovaries and in fat and bone in both genders. Ngf is highly expressed in reproductive tissues and p75NTR mRNA levels are respectively 300%, 100% and 50% higher in testis/ovaries/uterus than in brain. In contrast, BDNF genes are not expressed in reproductive tissues. As expected, Gprc6a is expressed in testis but not in the ovaries/uterus. A significant correlation was found between the expression levels of the gene ligands and their receptors in brain, BAT and testis suggesting a common pathway of different genes in these tissues in either male and female. Changes in the expression levels of osteocalcin, Ngf or Bdnf genes may mutually affect the expression levels of the others. Moreover, it may be possible that different ligands may operate through different receptor subtypes. Oxt and Oxtr failed to show significant correlation. The up-regulation of Ngf/p75NTR in BAT is

  5. Nerve Growth Factor, Brain-Derived Neurotrophic Factor and Osteocalcin Gene Relationship in Energy Regulation, Bone Homeostasis and Reproductive Organs Analyzed by mRNA Quantitative Evaluation and Linear Correlation Analysis.

    Science.gov (United States)

    Camerino, Claudia; Conte, Elena; Cannone, Maria; Caloiero, Roberta; Fonzino, Adriano; Tricarico, Domenico

    2016-01-01

    Nerve Growth Factor (NGF)/Brain-derived Neurotrophic Factor (BDNF) and osteocalcin share common effects regulating energy, bone mass, reproduction and neuronal functions. To investigate on the gene-relationship between NGF, BDNF, and Osteocalcin we compared by RT-PCR the transcript levels of Ngf, Bdnf and Osteocalcin as well as of their receptors p75NTR/NTRK1, NTRK2, and Gprc6a in brain, bone, white/brown adipose tissue (WAT/BAT) and reproductive organs of 3 months old female and male mice. Brain and bone were used as positive controls for NGF/BDNF and Osteocalcin respectively. The role of oxitocin(Oxt) and its receptor(Oxtr) was also investigated. Ngf expression shows an opposite trend compared to Bdnf. Ngf /p75NTR expression is 50% higher in BAT than brain, in both genders, but lower in bone. In contrast, Bdnf expression in bone is higher than in brain, but low in BAT/WAT. We found Osteocalcin gene expressed in brain in both genders, but Gprc6a expression is low in brain and BAT/WAT. As expected, Gprc6a gene is expressed in bone. Oxt gene was markedly expressed in brain, Oxtr in the ovaries and in fat and bone in both genders. Ngf is highly expressed in reproductive tissues and p75NTR mRNA levels are respectively 300, 100, and 50% higher in testis/ovaries/uterus than in brain. In contrast, BDNF genes are not expressed in reproductive tissues. As expected, Gprc6a is expressed in testis but not in the ovaries/uterus. A significant correlation was found between the expression levels of the gene ligands and their receptors in brain, BAT and testis suggesting a common pathway of different genes in these tissues in either male and female. Changes in the expression levels of osteocalcin, Ngf, or Bdnf genes may mutually affect the expression levels of the others. Moreover, it may be possible that different ligands may operate through different receptor subtypes. Oxt and Oxtr failed to show significant correlation. The up-regulation of Ngf /p75NTR in BAT is consistent

  6. Towards regulation of similar biotherapeutic products: Thailand's perspective.

    Science.gov (United States)

    Thanaphollert, Prapassorn; Tungsanga, Kriang

    2011-09-01

    The implementation of universal health coverage scheme in Thailand allows quality, equitable and accessible health care for all. Patients with life threatening and chronic diseases can get access to biotherapeutic products to treat their ailments. This triggered a major impact on the need for specific guidelines in evaluation of similar biotherapeutic products in order to standardize the regulatory pathway to license this class of products ensuring that the products meet acceptable levels of quality, safety and efficacy. The development of similar biotherapeutic products (SBP) should be considered to ensure therapeutic equivalence of biotherapeutics products at more affordable prices. This will lead to greater ease and speed of approval and assurance of the quality, safety and efficacy of these products. Therefore, we report herein the SBP situation in Thailand. Copyright © 2011. Published by Elsevier Ltd.

  7. Functional Integration of mRNA Translational Control Programs

    Directory of Open Access Journals (Sweden)

    Melanie C. MacNicol

    2015-07-01

    Full Text Available Regulated mRNA translation plays a key role in control of cell cycle progression in a variety of physiological and pathological processes, including in the self-renewal and survival of stem cells and cancer stem cells. While targeting mRNA translation presents an attractive strategy for control of aberrant cell cycle progression, mRNA translation is an underdeveloped therapeutic target. Regulated mRNAs are typically controlled through interaction with multiple RNA binding proteins (RBPs but the mechanisms by which the functions of distinct RBPs bound to a common target mRNA are coordinated are poorly understood. The challenge now is to gain insight into these mechanisms of coordination and to identify the molecular mediators that integrate multiple, often conflicting, inputs. A first step includes the identification of altered mRNA ribonucleoprotein complex components that assemble on mRNAs bound by multiple, distinct RBPs compared to those recruited by individual RBPs. This review builds upon our knowledge of combinatorial control of mRNA translation during the maturation of oocytes from Xenopus laevis, to address molecular strategies that may mediate RBP diplomacy and conflict resolution for coordinated control of mRNA translational output. Continued study of regulated ribonucleoprotein complex dynamics promises valuable new insights into mRNA translational control and may suggest novel therapeutic strategies for the treatment of disease.

  8. Growth Regulator Herbicides Prevent Invasive Annual Grass Seed Production

    Science.gov (United States)

    Auxinic herbicides, such as 2,4-D and dicamba, that act as plant growth regulators are commonly used for broadleaf weed control in cereal crops (e.g. wheat, barley), grasslands, and non-croplands. If applied at later growth stages, while cereals are developing reproductive parts, the herbicides can...

  9. Premarket Regulation of Tissue Engineered Medical Products in China.

    Science.gov (United States)

    Wang, Chunren; Wang, Anqi; Feng, Xiaoming; Ke, Linnan; Huang, Yuanli; Han, Qianqian; Sun, Xue; Yang, Zhaopeng

    2015-12-01

    Tissue engineered medical products (TEMPs) use state-of-the-art technologies and offer the patients with alternative clinical options for diseases that conventional treatments may fail or be incompetent. However promising, this technology is comparatively new with very limited hands-on experiences with both manufacturing and clinical therapy. Of great significance to products with such complexity and novelty is the establishment of a complete jurisdiction framework and a standardization database so that the safety of the technique in clinical treatment can be ensured. Although different regulatory routes are adopted in different countries, risks are generally considered to be derived from the cellular components within the product, the material scaffolds, and potentially from the final products. This article is to provide an insight of the regulatory considerations and the role of China Food and Drug Administration (CFDA) in the supervision of TEMPs.

  10. Regulation for Optimal Liquid Products during Biomass Pyrolysis: A Review

    Science.gov (United States)

    Wang, F.; Hu, L. J.; Zheng, Y. W.; Huang, Y. B.; Yang, X. Q.; Liu, C.; Kang, J.; Zheng, Z. F.

    2016-08-01

    The liquid product obtained from biomass pyrolysis is very valuable that it could be used for extraction of chemicals as well as for liquid fuel. The desire goal is to obtain the most bio-oil with desired higher heating value (HHV), high physicochemical stability. The yields and chemical composition of products from biomass pyrolysis are closely related to the feedstock, pyrolysis parameters and catalysts. Current researches mainly concentrated on the co-pyrolysis of different biomass and introduce of novel catalysts as well as the combined effect of catalysts and pyrolysis parameters. This review starts with the chemical composition of biomass and the fundamental parameters and focuses on the influence of catalysts on bio-oil. What is more, the pyrolysis facilities at commercial scales were also involved. The classic researches and the current literature about the yield and composition of products (mainly liquid products) are summarized.

  11. Metabolic Regulation of Natural Killer Cell IFN-? Production

    OpenAIRE

    Mah, Annelise Y.; Cooper, Megan A.

    2016-01-01

    Metabolism is critical for a host of cellular functions and provides a source of intracellular energy. It has been recognized recently that metabolism also regulates differentiation and effector functions of immune cells. Although initial work in this field has focused largely on T lymphocytes, recent studies have demonstrated metabolic control of innate immune cells, including natural killer (NK) cells. Here, we review what is known regarding the metabolic requirements for NK cell activation...

  12. Modulation of IFNAR1 mRNA expression in multiple sclerosis patients.

    Science.gov (United States)

    Serana, Federico; Sottini, Alessandra; Ghidini, Claudia; Zanotti, Cinzia; Capra, Ruggero; Cordioli, Cinzia; Caimi, Luigi; Imberti, Luisa

    2008-06-15

    Interferon-beta receptor (IFNAR) is composed of 2 subunits, IFNAR1 and IFNAR2, the latter of which is expressed as functional (IFNAR2.2), non-functional (IFNAR2.1) and soluble (IFNAR2.3) isoform. Real-Time PCR analysis of mRNA for all IFNAR components in multiple sclerosis patients naïve for therapy and undergoing long-term treatment with interferon-beta shows that IFNAR1 mRNA level is lower than in healthy controls. If long-term treated patients are divided according to the production of mRNA for Myxovirus protein-A, a marker of interferon-beta bioactivity, IFNAR1 mRNA reaches the values observed in controls only in Myxovirus protein-A-induced patients. Since chronic cell stimulation by interferon-beta induces IFNAR protein down-regulation, we suggest that the increase of IFNAR1 mRNA might serve as a mechanism for counterbalancing the loss of protein receptor, enhancing, at least in this sub-group of patients, cell responsiveness to interferon-beta.

  13. ALTERACIONES EN LA PRODUCCIÓN mRNA DE ENZIMAS INTESTINALES DE CERDOS DURANTE VARIOS PERÍODOS POSDESTETE ALTERAÇÕES NA PRODUÇÃO DE mRNA DE ENZIMAS INTESTINAIS DOS SUINOS DURANTE DIVERSOS PERÍODOS APOS O DESMAME ALTERATIONS IN THE PRODUCTION mRNA OF INTESTINAL ENZYMES IN PIGS DURING SEVERAL POSTWEANING PERIODS

    Directory of Open Access Journals (Sweden)

    CAROLINA MONTOYA R.

    2012-12-01

    enzimas ao nível do enterocito, causando a diminuição da absorção intestinal dos nutrientes, e provavelmente a apresentação do síndrome da diarreia apos o desmame.The aim of this study was to determine the gene expression of enzymes to enterocyte level of pigs during post-weaning periods.The experiment was conducted in San PabloProductionCenter of the Universidad Nacional de Colombia (Medellín with 16 weaned pigs at 21 days of age.The animals were fed for 10 days with a basal diet with milk and some of its derivatives, and that also fulfilled all the nutritionals minimums. Pigs were sequentially slaughtered on days one, five, seven, and 10 days after weaning.Complete extraction of small intestine was realized, which was divided into three sections (duodenum, jejunum and ileum. The gene expression of digestive enzymes by RT-PCR was evaluated.The statistical design used was completely at random. For intestinal enzymes significant decreases were showed, where on fifth day post-weaning presented the lowest values (P<0.01. Between one and 10 days post-weaning there were differences (P<0.01. The duodenum showed the highest values of gene expression (P<0.01. Early weaning alters molecular expression of enzymes to enterocyte level, causing the decrease of intestinal absorption of nutrients, and probably the presentation of post-weaning diarrhea syndrome.

  14. Production and regulation of lignin degrading enzymes from ...

    African Journals Online (AJOL)

    The importance of mycelia extension and enhanced enzyme production has biotechnological applications in wood and pulp, textile and tanning, as well as in oil industries. Key words: Basidiomycetes, Lentinus squarrosulus, Psathyrella atroumbonata, ligninase, cofactors. African Journal of Biotechnology Vol.2(11) 2003: ...

  15. New production regulates export stoichiometry in the ocean.

    Science.gov (United States)

    Tamelander, Tobias; Reigstad, Marit; Olli, Kalle; Slagstad, Dag; Wassmann, Paul

    2013-01-01

    The proportion in which carbon and growth-limiting nutrients are exported from the oceans' productive surface layer to the deep sea is a crucial parameter in models of the biological carbon pump. Based on >400 vertical flux observations of particulate organic carbon (POC) and nitrogen (PON) from the European Arctic Ocean we show the common assumption of constant C:N stoichiometry not to be met. Exported POC:PON ratios exceeded the classical Redfield atomic ratio of 6.625 in the entire region, with the largest deviation in the deep Central Arctic Ocean. In this part the mean exported POC:PON ratio of 9.7 (a:a) implies c. 40% higher carbon export compared to Redfield-based estimates. When spatially integrated, the potential POC export in the European Arctic was 10-30% higher than suggested by calculations based on constant POC:PON ratios. We further demonstrate that the exported POC:PON ratio varies regionally in relation to nitrate-based new production over geographical scales that range from the Arctic to the subtropics, being highest in the least productive oligotrophic Central Arctic Ocean and subtropical gyres. Accounting for variations in export stoichiometry among systems of different productivity will improve the ability of models to resolve regional patterns in carbon export and, hence, the oceans' contribution to the global carbon cycle will be predicted more accurately.

  16. New production regulates export stoichiometry in the ocean.

    Directory of Open Access Journals (Sweden)

    Tobias Tamelander

    Full Text Available The proportion in which carbon and growth-limiting nutrients are exported from the oceans' productive surface layer to the deep sea is a crucial parameter in models of the biological carbon pump. Based on >400 vertical flux observations of particulate organic carbon (POC and nitrogen (PON from the European Arctic Ocean we show the common assumption of constant C:N stoichiometry not to be met. Exported POC:PON ratios exceeded the classical Redfield atomic ratio of 6.625 in the entire region, with the largest deviation in the deep Central Arctic Ocean. In this part the mean exported POC:PON ratio of 9.7 (a:a implies c. 40% higher carbon export compared to Redfield-based estimates. When spatially integrated, the potential POC export in the European Arctic was 10-30% higher than suggested by calculations based on constant POC:PON ratios. We further demonstrate that the exported POC:PON ratio varies regionally in relation to nitrate-based new production over geographical scales that range from the Arctic to the subtropics, being highest in the least productive oligotrophic Central Arctic Ocean and subtropical gyres. Accounting for variations in export stoichiometry among systems of different productivity will improve the ability of models to resolve regional patterns in carbon export and, hence, the oceans' contribution to the global carbon cycle will be predicted more accurately.

  17. Effects of growth regulator herbicide on downy brome (Bromus tectorum) seed production

    Science.gov (United States)

    Previous research showed growth regulator herbicides, such as picloram and aminopyralid, have a sterilizing effect on Japanese brome (Bromus japonicus Thunb.) that can reduce this invasive annual grass’s seed production nearly 100%. This suggests growth regulators might be used to control invasive ...

  18. Laccases: Production, Expression Regulation, and Applications in Pharmaceutical Biodegradation

    Directory of Open Access Journals (Sweden)

    Jie Yang

    2017-05-01

    Full Text Available Laccases are a family of copper-containing oxidases with important applications in bioremediation and other various industrial and biotechnological areas. There have been over two dozen reviews on laccases since 2010 covering various aspects of this group of versatile enzymes, from their occurrence, biochemical properties, and expression to immobilization and applications. This review is not intended to be all-encompassing; instead, we highlighted some of the latest developments in basic and applied laccase research with an emphasis on laccase-mediated bioremediation of pharmaceuticals, especially antibiotics. Pharmaceuticals are a broad class of emerging organic contaminants that are recalcitrant and prevalent. The recent surge in the relevant literature justifies a short review on the topic. Since low laccase yields in natural and genetically modified hosts constitute a bottleneck to industrial-scale applications, we also accentuated a genus of laccase-producing white-rot fungi, Cerrena, and included a discussion with regards to regulation of laccase expression.

  19. DMPD: The atrial natriuretic peptide regulates the production of inflammatorymediators in macrophages. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 11890659 The atrial natriuretic peptide regulates the production of inflammatorymedia...tml) (.csml) Show The atrial natriuretic peptide regulates the production of inflammatorymediators in macrop...hages. PubmedID 11890659 Title The atrial natriuretic peptide regulates the production of inflammatorymedi

  20. Protein Kinase C-RegulatedProduction and Clearance

    Directory of Open Access Journals (Sweden)

    Taehyun Kim

    2011-01-01

    Full Text Available Alzheimer’s disease (AD is the most common form of dementia among the elderly population. AD, which is characterized as a disease of cognitive deficits, is mainly associated with an increase of amyloid β-peptide (Aβ in the brain. A growing body of recent studies suggests that protein kinase C (PKC promotes the production of the secretory form of amyloid precursor protein (sAPPα via the activation of α-secretase activity, which reduces the accumulation of pathogenic Aβ levels in the brain. Moreover, activation of PKCα and mitogen-activated protein kinase (MAPK is known to increase sAPPα. A novel type of PKC, PKCε, activates the Aβ degrading activity of endothelin converting enzyme type 1 (ECE-1, which might be mediated via the MAPK pathway as well. Furthermore, dysregulation of PKC-MAPK signaling is known to increase Aβ levels in the brain, which results in AD phenotypes. Here, we discuss roles of PKC in Aβ production and clearance and its implication in AD.

  1. Schisandra lignans production regulated by different bioreactor type.

    Science.gov (United States)

    Szopa, Agnieszka; Kokotkiewicz, Adam; Luczkiewicz, Maria; Ekiert, Halina

    2017-04-10

    Schisandra chinensis (Chinese magnolia vine) is a rich source of therapeutically relevant dibenzocyclooctadiene lignans with anticancer, immunostimulant and hepatoprotective activities. In this work, shoot cultures of S. chinensis were grown in different types of bioreactors with the aim to select a system suitable for the large scale in vitro production of schisandra lignans. The cultures were maintained in Murashige-Skoog (MS) medium supplemented with 3mg/l 6-benzylaminopurine (BA) and 1mg/l 1-naphthaleneacetic acid (NAA). Five bioreactors differing with respect to cultivation mode were tested: two liquid-phase systems (baloon-type bioreactor and bubble-column bioreactor with biomass immobilization), the gas-phase spray bioreactor and two commercially available temporary immersion systems: RITA(®) and Plantform. The experiments were run for 30 and 60 days in batch mode. The harvested shoots were evaluated for growth and lignan content determined by LC-DAD and LC-DAD-ESI-MS. Of the tested bioreactors, temporary immersion systems provided the best results with respect to biomass production and lignan accumulation: RITA(®) bioreactor yielded 17.86g/l (dry weight) during 60 day growth period whereas shoots grown for 30 days in Plantform bioreactor contained the highest amount of lignans (546.98mg/100g dry weight), with schisandrin, deoxyschisandrin and gomisin A as the major constituents (118.59, 77.66 and 67.86mg/100g dry weight, respectively). Copyright © 2017 Elsevier B.V. All rights reserved.

  2. MCPIP-1, Alias Regnase-1, Controls Epithelial Inflammation by Posttranscriptional Regulation of IL-8 Production.

    Science.gov (United States)

    Dobosz, Ewelina; Wilamowski, Mateusz; Lech, Maciej; Bugara, Beata; Jura, Jolanta; Potempa, Jan; Koziel, Joanna

    2016-01-01

    Pattern recognition receptors are critical for the detection of invading microorganisms. They activate multiple pathways that lead to the induction of proinflammatory responses and pathogen clearance. The intensity and duration of this immune reaction must be tightly controlled spatially and temporally in every tissue by different negative regulators. We hypothesized that monocyte chemoattractant protein-1-induced protein-1 (MCPIP-1) might play a role in maintaining immune homeostasis in the epithelium both under physiological conditions and upon bacterial infection. To this end, we examined the distribution of the MCPIP-1 transcript and protein in various tissues. The MCPIP-1 protein level was higher in epithelial cells than in myeloid cells. MCPIP-1 exerted RNase activity towards the interleukin (IL)-8 transcript and the lifespan of IL-8 was determined by the presence of the stem-loops/hairpin structures at the 3'UTR region of IL-8 mRNA. Moreover, using fully active, purified recombinant MCPIP-1 protein, we elucidated the mechanism by which MCPIP-1 controls the IL-8 mRNA level. In conclusion, we uncovered a novel IL-8-dependent mechanism via which MCPIP-1 maintains epithelial homeostasis. This study reveals for the first time that MCPIP-1 plays a crucial anti-inflammatory role not only in myeloid cells but also in epithelial cells. © 2016 S. Karger AG, Basel.

  3. The importance of product definitions in US e-cigarette laws and regulations.

    Science.gov (United States)

    Lempert, Lauren K; Grana, Rachel; Glantz, Stanton A

    2016-04-01

    How electronic cigarettes and similar products (e-cigarettes) are defined affects how they are regulated, particularly whether existing laws for cigarettes apply, including sales and marketing, youth access, smoke-free and taxation laws. We examined the text of 46 bills that define e-cigarettes enacted in 40 states and characterised how e-cigarettes and similar products were defined. States enact laws creating new product categories for e-cigarettes separate from the 'tobacco product' category (eg, 'alternative nicotine product,' 'vapour product,' 'electronic nicotine device'), with four states explicitly excluding e-cigarettes from 'tobacco products.' Twenty-eight states do not include e-cigarettes in their definitions of 'tobacco products' or 'smoking,' eight include e-cigarettes as 'tobacco products,' three include e-cigarettes in 'smoking.' Sixteen states' definitions of e-cigarettes require nicotine, and five states pre-empt more stringent local laws. Tobacco and e-cigarette industry representatives tried to shape laws that benefit their interests. Definitions separating e-cigarettes from other tobacco products are common. Similar to past 'Trojan horse' policies, e-cigarette policies that initially appear to restrict sales (eg, limit youth access) may actually undermine regulation if they establish local pre-emption or create definitions that divide e-cigarettes from other tobacco products. Comparable issues are raised by the European Union Tobacco Products Directive and e-cigarette regulations in other countries. Policymakers should carefully draft legislation with definitions of e-cigarettes that broadly define the products, do not require nicotine or tobacco, do not pre-empt stronger regulations and explicitly include e-cigarettes in smoke-free and taxation laws. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  4. Human-derived gut microbiota modulates colonic secretion in mice by regulating 5-HT3receptor expression via acetate production.

    Science.gov (United States)

    Bhattarai, Yogesh; Schmidt, Bradley A; Linden, David R; Larson, Eric D; Grover, Madhusudan; Beyder, Arthur; Farrugia, Gianrico; Kashyap, Purna C

    2017-07-01

    Serotonin [5-hydroxytryptamine (5-HT)], an important neurotransmitter and a paracrine messenger in the gastrointestinal tract, regulates intestinal secretion by its action primarily on 5-HT 3 and 5-HT 4 receptors. Recent studies highlight the role of gut microbiota in 5-HT biosynthesis. In this study, we determine whether human-derived gut microbiota affects host secretory response to 5-HT and 5-HT receptor expression. We used proximal colonic mucosa-submucosa preparation from age-matched Swiss Webster germ-free (GF) and humanized (HM; ex-GF colonized with human gut microbiota) mice. 5-HT evoked a significantly greater increase in short-circuit current (Δ I sc ) in GF compared with HM mice. Additionally, 5-HT 3 receptor mRNA and protein expression was significantly higher in GF compared with HM mice. Ondansetron, a 5-HT 3 receptor antagonist, inhibited 5-HT-evoked Δ I sc in GF mice but not in HM mice. Furthermore, a 5-HT 3 receptor-selective agonist, 2-methyl-5-hydroxytryptamine hydrochloride, evoked a significantly higher Δ I sc in GF compared with HM mice. Immunohistochemistry in 5-HT 3A -green fluorescent protein mice localized 5-HT 3 receptor expression to enterochromaffin cells in addition to nerve fibers. The significant difference in 5-HT-evoked Δ I sc between GF and HM mice persisted in the presence of tetrodotoxin (TTX) but was lost after ondansetron application in the presence of TTX. Application of acetate (10 mM) significantly lowered 5-HT 3 receptor mRNA in GF mouse colonoids. We conclude that host secretory response to 5-HT may be modulated by gut microbiota regulation of 5-HT 3 receptor expression via acetate production. Epithelial 5-HT 3 receptor may function as a mediator of gut microbiota-driven change in intestinal secretion. NEW & NOTEWORTHY We found that gut microbiota alters serotonin (5-HT)-evoked intestinal secretion in a 5-HT 3 receptor-dependent mechanism and gut microbiota metabolite acetate alters 5-HT 3 receptor expression in

  5. Children's Development of Self-Regulation in Speech Production

    DEFF Research Database (Denmark)

    MacDonald, Ewen; Johnson, Elizabeth K.; Forsythe, Jaime

    2012-01-01

    , fall into this latter class of vocal learning. Self-monitoring has been assumed to play an important role in the vocal learning of speech [1–3] and studies demonstrate that perception of your own voice is crucial for both the development and lifelong maintenance of vocalizations in humans and songbirds...... [4–8]. Experimental modifications of auditory feedback can also change vocalizations in both humans and songbirds [9–13]. However, with the exception of large manipulations of timing [14, 15], no study to date has ever directly examined the use of auditory feedback in speech production under the age...... of 4. Here we use a real-time formant perturbation task [16] to compare the response of toddlers, children, and adults to altered feedback. Children and adults reacted to this manipulation by changing their vowels in a direction opposite to the perturbation. Surprisingly, toddlers' speech didn't change...

  6. Scientific problems in the regulation of red blood cell products.

    Science.gov (United States)

    Hess, John R

    2012-08-01

    For the past 30 years, red blood cell (RBC) storage systems have been licensed in the United States based on the demonstration that 24-hour in vivo recovery was greater than 75% and hemolysis was less than 1%. Now additional requirements for storage system licensure have being added. The meaning and value of these new requirements have been questioned. The literature regarding the performance of present and suggested new tests for RBC licensure was reviewed. (51) Cr 24-hr in vivo recovery has an intrinsic 4% error of measurement whereas the error in measures of hemolysis is less than 0.1%. Both measures have large donor-dependent end-of-storage variability; nevertheless, they have successfully guided RBC storage system development for six decades. Adenosine 5'-triphosphate and 2,3-diphosphoglycerate are difficult to measure accurately and international shared-sample studies suggest 6 and 11% coefficients of variation across laboratories. There is no readily available way to measure the oxygen equilibrium curve accurately. The new failure criteria provide no useful information and randomly fail good products. Attempts to expand the useful regulatory requirements for RBC storage system licensure are limited by poor understanding of the storage lesion and its effect of RBC performance. Measures of (51) Cr 24-hour in vivo recovery remain critical and resources for this measure are limiting. The interaction between limited testing resources and large donor variability remains a major limit on RBC storage system development. It is important that new required tests contribute meaningful information and not make development and licensure of better products more difficult. © 2012 American Association of Blood Banks.

  7. Endothelin Regulates Porphyromonas gingivalis-Induced Production of Inflammatory Cytokines.

    Directory of Open Access Journals (Sweden)

    Ga-Yeon Son

    Full Text Available Periodontitis is a very common oral inflammatory disease that results in the destruction of supporting connective and osseous tissues of the teeth. Although the exact etiology is still unclear, Gram-negative bacteria, especially Porphyromonas gingivalis in subgingival pockets are thought to be one of the major etiologic agents of periodontitis. Endothelin (ET is a family of three 21-amino acid peptides, ET-1, -2, and -3, that activate G protein-coupled receptors, ETA and ETB. Endothelin is involved in the occurrence and progression of various inflammatory diseases. Previous reports have shown that ET-1 and its receptors, ETA and ETB are expressed in the periodontal tissues and, that ET-1 levels in gingival crevicular fluid are increased in periodontitis patients. Moreover, P. gingivalis infection has been shown to induce the production of ET-1 along with other inflammatory cytokines. Despite these studies, however, the functional significance of endothelin in periodontitis is still largely unknown. In this study, we explored the cellular and molecular mechanisms of ET-1 action in periodontitis using human gingival epithelial cells (HGECs. ET-1 and ETA, but not ETB, were abundantly expressed in HGECs. Stimulation of HGECs with P. gingivalis or P. gingivalis lipopolysaccharide increased the expression of ET-1 and ETA suggesting the activation of the endothelin signaling pathway. Production of inflammatory cytokines, IL-1β, TNFα, and IL-6, was significantly enhanced by exogenous ET-1 treatment, and this effect depended on the mitogen-activated protein kinases via intracellular Ca2+ increase, which resulted from the activation of the phospholipase C/inositol 1,4,5-trisphosphate pathway. The inhibition of the endothelin receptor-mediated signaling pathway with the dual receptor inhibitor, bosentan, partially ameliorated alveolar bone loss and immune cell infiltration. These results suggest that endothelin plays an important role in P. gingivalis

  8. Encountering Challenges with the EU Regulation on Advance Therapy Medical Products.

    Science.gov (United States)

    Mansnérus, Juli

    2015-12-01

    This article aims at analysing how well the Advanced Therapy Medical Product Regulation (EC) No. 1394/2007 (ATMP Regulation) meets the needs of small and medium-sized enterprises (SMES), academia and public tissue establishments developing advanced therapy medical products (ATMPS). Benefits and shortcomings of the ATMP Regulation are identified, and possible amendments are proposed to accelerate the translation of research into advanced therapies and to facilitate the commercialisation of ATMPS whilst ensuring safety. It was set up as a lex specialis to ensure the free movement of ATMPS within the EU in order to facilitate their access to the internal market and to foster the competitiveness of European pharmaceutical companies, while guaranteeing the highest level protection of public health. Since the adoption of the ATMP Regulation in late 2008, only 5 ATMPS have been granted marketing authorisations thus far. Hence, there is a need to analyse whether the ATMP Regulation meets its objectives.

  9. Applying the breaks on gene expression - mRNA deadenylation by Pop2p

    DEFF Research Database (Denmark)

    Andersen, Kasper Røjkjær; Jonstrup, Anette Thyssen; Van, Lan Bich

    When driving a car, control of the brakes is just as important as control of the accelerator pedal. Likewise, in gene expression, regulation of mRNA degradation is as important as regulation of its synthesis (Mühlemann, 2005). The rate-determining step of mRNA decay in eukaryotes seems to be the ......When driving a car, control of the brakes is just as important as control of the accelerator pedal. Likewise, in gene expression, regulation of mRNA degradation is as important as regulation of its synthesis (Mühlemann, 2005). The rate-determining step of mRNA decay in eukaryotes seems...

  10. Regulations applicable to plant food supplements and related products in the European Union.

    Science.gov (United States)

    Silano, Vittorio; Coppens, Patrick; Larrañaga-Guetaria, Ainhoa; Minghetti, Paola; Roth-Ehrang, René

    2011-12-01

    This paper deals with the current regulatory and legal settings of traditional plant food supplements and herbal medicinal products in the European Union (EU). Marketing of botanicals in foods and food supplements in the EU is subject to several provisions of food law, which cover aspects of safety, production, labelling and product composition, including the use of additives and maximum levels of contaminants and residues. However, due to limited harmonization at the EU level, specific national regulations adopted at a Member State level also apply and mutual recognition is the mechanism through which such products can be marketed in EU countries other than those of origin. Unlike food supplements, marketing of traditional herbal medicinal products is regulated by an ad hoc Directive (i.e. Directive 2004/24/EC) covering in detail all the relevant aspects of these products, including a facilitated registration procedure at national level. However, by distinguishing traditional herbal medicinal products from plant food supplements and establishing selective marketing modalities for these two product categories, the EU has been confronted with implementation difficulties for traditional herbal medicinal products and a lack of homogeneity in the regulatory approaches adopted in different EU Member States. In fact, currently the nature of the commercial botanical products made available to consumers as traditional medicinal products or food supplements, depends largely on the EU Member State under consideration as a consequence of how competent National Authorities and manufacturing companies interpret and apply current regulations rather than on the intrinsic properties of the botanical products and their constituents. When the EU approach is compared with approaches adopted in some non-European countries to regulate these product categories, major differences become evident.

  11. CdtR Regulates TcdA and TcdB Production in Clostridium difficile.

    Directory of Open Access Journals (Sweden)

    Shelley A Lyon

    2016-07-01

    Full Text Available Clostridium difficile is a global health burden and the leading cause of antibiotic-associated diarrhoea worldwide, causing severe gastrointestinal disease and death. Three well characterised toxins are encoded by this bacterium in two genetic loci, specifically, TcdB (toxin B and TcdA (toxin A in the Pathogenicity Locus (PaLoc and binary toxin (CDT in the genomically distinct CDT locus (CdtLoc. Toxin production is controlled by regulators specific to each locus. The orphan response regulator, CdtR, encoded within the CdtLoc, up-regulates CDT production. Until now there has been no suggestion that CdtR influences TcdA and TcdB production since it is not carried by all PaLoc-containing strains and CdtLoc is not linked genetically to PaLoc. Here we show that, in addition to CDT, CdtR regulates TcdA and TcdB production but that this effect is strain dependent. Of clinical relevance, CdtR increased the production of TcdA, TcdB and CDT in two epidemic ribotype 027 human strains, modulating their virulence in a mouse infection model. Strains traditionally from animal lineages, notably ribotype 078 strains, are increasingly being isolated from humans and their genetic and phenotypic analysis is critical for future studies on this important pathogen. Here we show that CdtR-mediated toxin regulation did not occur in other strain backgrounds, including a ribotype 078 animal strain. The finding that toxin gene regulation is strain dependent highlights the regulatory diversity between C. difficile isolates and the importance of studying virulence regulation in diverse lineages and clinically relevant strains. Our work provides the first evidence that TcdA, TcdB and CDT production is linked by a common regulatory mechanism and that CdtR may act as a global regulator of virulence in epidemic 027 strains.

  12. Expression of brain derived neurotrophic factor, activity-regulated cytoskeleton protein mRNA, and enhancement of adult hippocampal neurogenesis in rats after sub-chronic and chronic treatment with the triple monoamine re-uptake inhibitor tesofensine

    DEFF Research Database (Denmark)

    Larsen, Marianne Hald; Rosenbrock, Holger; Sams-Dodd, Frank

    2007-01-01

    D-immunoreactivity. We find that chronic, but not sub-chronic treatment with Tesofensine increases BDNF mRNA in the CA3 region of the hippocampus (35%), and Arc mRNA in the CA1 of the hippocampus (65%). Furthermore, the number of Ki-67- and neuroD-positive cells increased after chronic, but not sub-chronic treatment....... This study shows that Tesofensine enhances hippocampal gene expression and new cell formation indicative for an antidepressant potential of this novel drug substance....

  13. Influence of pH Regulation Mode in Glucose Fermentation on Product Selection and Process Stability

    OpenAIRE

    Zuhaida Mohd-Zaki; Juan R. Bastidas-Oyanedel; Yang Lu; Robert Hoelzle; Steven Pratt; Fran R. Slater; Batstone, Damien J.

    2016-01-01

    Mixed culture anaerobic fermentation generates a wide range of products from simple sugars, and is potentially an effective process for producing renewable commodity chemicals. However it is difficult to predict product spectrum, and to control the process. One of the key control handles is pH, but the response is commonly dependent on culture history. In this work, we assess the impact of pH regulation mode on the product spectrum. Two regulation modes were applied: in the first, pH was adju...

  14. Increase in IL-8, IL-10, IL-13, and RANTES mRNA levels (in situ hybridization) in the nasal mucosa after nasal allergen provocation

    NARCIS (Netherlands)

    Kleinjan, A.; Dijkstra, M. D.; Boks, S. S.; Severijnen, L. A.; Mulder, P. G.; Fokkens, W. J.

    1999-01-01

    BACKGROUND: Allergic inflammation is regulated by the local production and release of several cytokines. OBJECTIVES: This study was designed to assess the changes in mRNA cytokine-positive cells after allergen provocation and to compare these cytokines with tissue eosinophilia as a marker of

  15. Up-regulation of avian uncoupling protein in cold-acclimated and hyperthyroid ducklings prevents reactive oxygen species production by skeletal muscle mitochondria.

    Science.gov (United States)

    Rey, Benjamin; Roussel, Damien; Romestaing, Caroline; Belouze, Maud; Rouanet, Jean-Louis; Desplanches, Dominique; Sibille, Brigitte; Servais, Stéphane; Duchamp, Claude

    2010-04-28

    Although identified in several bird species, the biological role of the avian homolog of mammalian uncoupling proteins (avUCP) remains extensively debated. In the present study, the functional properties of isolated mitochondria were examined in physiological or pharmacological situations that induce large changes in avUCP expression in duckling skeletal muscle. The abundance of avUCP mRNA, as detected by RT-PCR in gastrocnemius muscle but not in the liver, was markedly increased by cold acclimation (CA) or pharmacological hyperthyroidism but was down-regulated by hypothyroidism. Activators of UCPs, such as superoxide with low doses of fatty acids, stimulated a GDP-sensitive proton conductance across the inner membrane of muscle mitochondria from CA or hyperthyroid ducklings. The stimulation was much weaker in controls and not observed in hypothyroid ducklings or in any liver mitochondrial preparations. The production of endogenous mitochondrial reactive oxygen species (ROS) was much lower in muscle mitochondria from CA and hyperthyroid ducklings than in the control or hypothyroid groups. The addition of GDP markedly increased the mitochondrial ROS production of CA or hyperthyroid birds up to, or above, the level of control or hypothyroid ducklings. Differences in ROS production among groups could not be attributed to changes in antioxidant enzyme activities (superoxide dismutase or glutathione peroxidase). This work provides the first functional in vitro evidence that avian UCP regulates mitochondrial ROS production in situations of enhanced metabolic activity.

  16. Up-regulation of avian uncoupling protein in cold-acclimated and hyperthyroid ducklings prevents reactive oxygen species production by skeletal muscle mitochondria

    Directory of Open Access Journals (Sweden)

    Servais Stéphane

    2010-04-01

    Full Text Available Abstract Background Although identified in several bird species, the biological role of the avian homolog of mammalian uncoupling proteins (avUCP remains extensively debated. In the present study, the functional properties of isolated mitochondria were examined in physiological or pharmacological situations that induce large changes in avUCP expression in duckling skeletal muscle. Results The abundance of avUCP mRNA, as detected by RT-PCR in gastrocnemius muscle but not in the liver, was markedly increased by cold acclimation (CA or pharmacological hyperthyroidism but was down-regulated by hypothyroidism. Activators of UCPs, such as superoxide with low doses of fatty acids, stimulated a GDP-sensitive proton conductance across the inner membrane of muscle mitochondria from CA or hyperthyroid ducklings. The stimulation was much weaker in controls and not observed in hypothyroid ducklings or in any liver mitochondrial preparations. The production of endogenous mitochondrial reactive oxygen species (ROS was much lower in muscle mitochondria from CA and hyperthyroid ducklings than in the control or hypothyroid groups. The addition of GDP markedly increased the mitochondrial ROS production of CA or hyperthyroid birds up to, or above, the level of control or hypothyroid ducklings. Differences in ROS production among groups could not be attributed to changes in antioxidant enzyme activities (superoxide dismutase or glutathione peroxidase. Conclusion This work provides the first functional in vitro evidence that avian UCP regulates mitochondrial ROS production in situations of enhanced metabolic activity.

  17. The global regulator LaeA controls production of citric acid and endoglucanases in Aspergillus carbonarius.

    Science.gov (United States)

    Linde, Tore; Zoglowek, Marta; Lübeck, Mette; Frisvad, Jens Christian; Lübeck, Peter Stephensen

    2016-08-01

    The global regulatory protein LaeA is known for regulating the production of many kinds of secondary metabolites in Aspergillus species, as well as sexual and asexual reproduction, and morphology. In Aspergillus carbonarius, it has been shown that LaeA regulates production of ochratoxin. We have investigated the regulatory effect of LaeA on production of citric acid and cellulolytic enzymes in A. carbonarius. Two types of A. carbonarius strains, having laeA knocked out or overexpressed, were constructed and tested in fermentation. The knockout of laeA significantly decreased the production of citric acid and endoglucanases, but did not reduce the production of beta-glucosidases or xylanases. The citric acid accumulation was reduced with 74-96 % compared to the wild type. The endoglucanase activity was reduced with 51-78 %. Overexpression of LaeA seemed not to have an effect on citric acid production or on cellulose or xylanase activity.

  18. Carboxymethyl-cellulase from Erwina chrysanthemi. I. Production and regulation of extracellular carboxymethyl-cellulase

    Energy Technology Data Exchange (ETDEWEB)

    Boyer, M.H.; Chambost, J.P.; Magnan, M.; Cattaneo, J.

    1984-01-01

    Erwinia chrysanthemi strain 3665 growing aerobically in a mineral salts medium containing various carbon sources constitutively secreted low levels of carboxymethyl-cellulase activity. Increased production of this activity was triggered by conditions which reduced the growth rate. The results obtained with continuous culture suggested that this production was controlled by a mechanism similar to catabolite repression. However, other factors might be implicated in the regulation of cellulase production.

  19. Role of mRNA Methylation in Prostate Cancer

    Science.gov (United States)

    2015-02-01

    regulation of mRNA. Recent technological advances have made it possible to detect mRNA methylation . The m6A was found near regulatory regions and...TERMS mRNA methylation , FTO, MeRIP-seq, RNA-seq, m6A 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF...10 9. Appendices……………………………………………………………10 1. INTRODUCTION: Methylation at the N6 position of adenosine ( m6A ) is a post-transcriptional modification of

  20. Lipopolysaccharide-regulated production of bone sialoprotein and interleukin-8 in human periodontal ligament fibroblasts: the role of toll-like receptors 2 and 4 and the MAPK pathway.

    Science.gov (United States)

    Zhang, Y; Li, X

    2015-04-01

    Lipopolysaccharide (LPS) on the cell wall of periodontal pathogens is a major mediator of the inflammatory response and can enhance alveolar bone resorption in periodontitis. Bone sialoprotein is an early marker of osteoblast differentiation. The proinflammatory cytokine, interleukin-8 (IL-8), induces osteoclast differentiation, maturation and maintenance of bone resorption activity. However, the effects of LPS from periodontal pathogens on the expression of bone sialoprotein and IL-8 in human osteoblasts and the mechanism of periodontal bone metabolism regulation are rather unclear. The objectives of this study were to determine the effects of Porphyromonas gingivalis LPS on the production of bone sialoprotein and IL-8 in human periodontal ligament fibroblasts (hPDLFs), and to investigate whether toll-like receptor (TLR) 2, TLR4 and MAPKs pathways are involved in the regulation of production of bone sialoprotein and IL-8 by P. gingivalis LPS. The third-generation of hPDLFs were cultured with mineralization-inducing culture medium. After hPDLFs were treated with P. gingivalis LPS, bone sialoprotein and IL-8 mRNA expression were detected using Real time PCR. Then hPDLFs were transiently transfected with siTLR2 or siTLR4 (20 nm) or inhibited by MAPK signaling pathways inhibitors, and then bone sialoprotein and IL-8 mRNA and protein expression were also detected using Real time PCR and western blotting. Treatments with 0.01 and 0.1 mg/L of P. gingivalis LPS for 8 h up-regulated bone sialoprotein mRNA expression, whereas 10 and 100 mg/L of P. gingivalis LPS induced a significant decrease in the expression of bone sialoprotein mRNA. In contrast, IL8 mRNA levels were increased significantly by 10 mg/L of P. gingivalis LPS. Interestingly, small interfering RNA (siRNA) knock down of the TLR2 and ERK1/2 inhibitor, PD98059, abolished the effects of P. gingivalis LPS on the bone sialoprotein mRNA level, whereas siRNA knock down of the TLR2 and p38 MAPK inhibitor

  1. Can the FDA Keep Kosher?: Regulation of Kosher Claims on Product Labels

    OpenAIRE

    Gottlieb, Caren

    1998-01-01

    Product labels blast their message through the supermarket aisles, vying to attract the attention of consumers. However, the advertising means available to manufacturers are limited by laws that regulate the claims that they may make. Regulation of "kosher claims" on food packaging implicates many of these general labeling issues, as well as several unique problems involving freedom of religion. The presence of religious issues provides a stronger justification for State involvement, as well ...

  2. Management practices in Australasian ethical investment products: a role for regulation?

    DEFF Research Database (Denmark)

    Haigh, Matthew; Guthrie, James

    2010-01-01

    This paper adds to the literatures on socially responsible investment (SRI), investment management, regulation of financial services and social accounting by providing a comprehensive survey of investment methods used in SRI products and regulated social reporting in financial services. Australian...... a four-year period: 2004-2007. These aspects were further examined in 18 case studies. Over the period, diversity and intensity of construction methods had increased both within and between investment managers. The non-standard nature of management consultation used in SRI products, marketing needs...... to distinguish rather than standardize investment methods and the types of information thought relevant to clients did not reconcile easily with the types of information required by regulation. The more recent products in the sample tended to reference market indexes in portfolio construction, separate social...

  3. The feed-back regulation of erythropoietin production in healthy humans

    Energy Technology Data Exchange (ETDEWEB)

    Klausen, T

    1998-10-01

    The proposed oxygen-dependent feed-back loop regulation of EPO (erythropoietin) production is mainly supported by data from studies in animals and cell cultures. The feed-back loop and its dependence on oxygen was therefore challenged by studies in healthy humans: Exposure of humans to different levels of acute and continued altitude hypobaria provided evidence for an oxygen dependence of the EPO response. This response is consistent with the proposed feed-back loop regulation of EPO production; Exposure to continued altitude hypobaria demonstrated that the decline in human EPO production is initiated before an EPO-induced erythopoiesis is detectable, and that this decline is related to a concomitant decrease in the haemoglobin-oxygen affinity. Contrary to the feed-back loop, this time-relation indicate that the feed-back regulation of EPO production during continued hypobaric hypoxia is exerted primarily through a decrease in the haemoglobin-oxygen affinity, rather than by the effects of an EPO-stimulated erythropoiesis; Increased circulating levels of the proinflammatory cytokine IL-6 was found in healthy humans during four days of altitude exposure as compared with sea level. The other proinflammatory cytokines IL-1 beta, and TNF alpha remained unchanged, and the increased serum IL-6 did not induce production of c-reactive protein; Comparable circadian variations in human EPO production were shown in sedentary subjects, athletes, and healthy but hypoxaemic subjects. Human EPO production could not be triggered by one hour of high-intensity exercise, whereas longitudinal changes in exercise showed a trend of relation between human EPO production, serum concentration of free testosterone, and indices of body composition. These results have demonstrated and endogenous, probably hormonal, and oxygen-independent regulation of human EPO production, which is at variance with the oxygen dependent feed-back loop regulation of EPO production. Conclusively, the present

  4. Unfinished business in the regulation of shale gas production in the United States.

    Science.gov (United States)

    Centner, Terence J; O'Connell, Laura Kathryn

    2014-04-01

    With increased drilling for natural gas, toxic chemicals used to fracture wells have been introduced into the environment accompanied by allegations of injuries. This article evaluates laws and regulations governing shale gas production to disclose ideas for offering further protection to people and the environment. The aim of the study is to offer state governments ideas for addressing contractual obligations of drilling operators, discerning health risks, disclosing toxic chemicals, and reporting sufficient information to detect problems and enforce regulations. The discussion suggests opportunities for state regulators to become more supportive of public health through greater oversight of shale gas extraction. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Estrogens, selective estrogen receptor modulators, and a selective estrogen receptor down-regulator inhibit endothelial production of tissue factor pathway inhibitor 1

    Directory of Open Access Journals (Sweden)

    Ree Anne

    2006-10-01

    Full Text Available Abstract Background Hormone therapy, oral contraceptives, and tamoxifen increase the risk of thrombotic disease. These compounds also reduce plasma content of tissue factor pathway inhibitor-1 (TFPI, which is the physiological inhibitor of the tissue factor pathway of coagulation. The current aim was to study if estrogens and estrogen receptor (ER modulators may inhibit TFPI production in cultured endothelial cells and, if so, identify possible mechanisms involved. Methods Human endothelial cell cultures were treated with 17β-estradiol (E2, 17α-ethinylestradiol (EE2, tamoxifen, raloxifene, or fulvestrant. Protein levels of TFPI in cell media and cell lysates were measured by an enzyme-linked immunosorbent assay, and TFPI mRNA levels were assessed by quantitative PCR. Expression of ERα was analysed by immunostaining. Results All compounds (each in a concentration of 10 nM reduced TFPI in cell medium, by 34% (E2, 21% (EE2, 16% (tamoxifen, and 28% (raloxifene, respectively, with identical inhibitory effects on cellular TFPI levels. Expression of TFPI mRNA was principally unchanged. Treatment with fulvestrant, which was also associated with down-regulation of secreted TFPI (9% with 10 nM and 26% with 1000 nM, abolished the TFPI-inhibiting effect of raloxifene, but not of the other compounds. Notably, the combination of 1000 nM fulvestrant and 10 nM raloxifene increased TFPI secretion, and, conversely, 10 nM of either tamoxifen or raloxifene seemed to partly (tamoxifen or fully (raloxifene counteract the inhibitory effect of 1000 nM fulvestrant. The cells did not express the regular nuclear 66 kDa ERα, but instead a 45 kDa ERα, which was not regulated by estrogens or ER modulators. Conclusion E2, EE2, tamoxifen, raloxifene, and fulvestrant inhibited endothelial production of TFPI by a mechanism apparently independent of TFPI transcription.

  6. From stem cell to erythroblast: regulation of red cell production at multiple levels by multiple hormones.

    Science.gov (United States)

    Lodish, Harvey; Flygare, Johan; Chou, Song

    2010-07-01

    This article reviews the regulation of production of red blood cells at several levels: (1) the ability of erythropoietin and adhesion to a fibronectin matrix to stimulate the rapid production of red cells by inducing terminal proliferation and differentiation of committed erythroid CFU-E progenitors; (2) the regulated expansion of the pool of earlier BFU-E erythroid progenitors by glucocorticoids and other factors that occurs during chronic anemia or inflammation; and (3) the expansion of thehematopoietic cell pool to produce more progenitors of all hematopoietic lineages. (c) 2010 IUBMB IUBMB Life.

  7. Validation of reference genes for normalization of qPCR mRNA expression levels in Staphylococcus aureus exposed to osmotic and lactic acid stress conditions encountered during food production and preservation.

    Science.gov (United States)

    Sihto, Henna-Maria; Tasara, Taurai; Stephan, Roger; Johler, Sophia

    2014-07-01

    Staphylococcus aureus represents the most prevalent cause of food-borne intoxications worldwide. While being repressed by competing bacteria in most matrices, this pathogen exhibits crucial competitive advantages during growth at high salt concentrations or low pH, conditions frequently encountered in food production and preservation. We aimed to identify reference genes that could be used to normalize qPCR mRNA expression levels during growth of S. aureus in food-related osmotic (NaCl) and acidic (lactic acid) stress adaptation models. Expression stability of nine housekeeping genes was evaluated in full (LB) and nutrient-deficient (CYGP w/o glucose) medium under conditions of osmotic (4.5% NaCl) and acidic stress (lactic acid, pH 6.0) after 2-h exposure. Among the set of candidate reference genes investigated, rplD, rpoB,gyrB, and rho were most stably expressed in LB and thus represent the most suitable reference genes for normalization of qPCR data in osmotic or lactic acid stress models in a rich medium. Under nutrient-deficient conditions, expression of rho and rpoB was highly stable across all tested conditions. The presented comprehensive data on changes in expression of various S. aureus housekeeping genes under conditions of osmotic and lactic acid stress facilitate selection of reference genes for qPCR-based stress response models. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  8. Annexin A5 regulates Leydig cell testosterone production via ERK1/2 pathway.

    Science.gov (United States)

    He, Ze; Sun, Qin; Liang, Yuan-Jiao; Chen, Li; Ge, Yi-Feng; Yun, Shi-Feng; Yao, Bing

    2016-01-01

    This study was to investigate the effect of annexin A5 on testosterone secretion from primary rat Leydig cells and the underlying mechanisms. Isolated rat Leydig cells were treated with annexin A5. Testosterone production was detected by chemiluminescence assay. The protein and mRNA of Steroidogenic acute regulatory (StAR), P450scc, 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD), and 17α-hydroxylase were examined by Western blotting and semi-quantitative RT-PCR, respectively. Annexin A5 significantly stimulated testosterone secretion from rat Leydig cells in dose- and time-dependent manners and increased mRNA and protein expression of StAR, P450scc, 3β-HSD, and 17β-HSD but not 17α-hydroxylase. Annexin A5 knockdown by siRNA significantly decreased the level of testosterone and protein expression of P450scc, 3β-HSD, and 17β-HSD. The significant activation of ERK1/2 signaling was observed at 5, 10, and 30 min after annexin A5 treatment. After the pretreatment of Leydig cells with ERK inhibitor PD98059 (50 μmol l-1 ) for 20 min, the effects of annexin A5 on promoting testosterone secretion and increasing the expression of P450scc, 3β-HSD, and 17β-HSD were completely abrogated (P testosterone production and the expression of P450scc, 3β-HSD, and 17β-HSD in Leydig cells.

  9. Annexin A5 regulates Leydig cell testosterone production via ERK1/2 pathway

    Directory of Open Access Journals (Sweden)

    Ze He

    2016-01-01

    Full Text Available This study was to investigate the effect of annexin A5 on testosterone secretion from primary rat Leydig cells and the underlying mechanisms. Isolated rat Leydig cells were treated with annexin A5. Testosterone production was detected by chemiluminescence assay. The protein and mRNA of Steroidogenic acute regulatory (StAR, P450scc, 3β-hydroxysteroid dehydrogenase (3β-HSD, 17β-hydroxysteroid dehydrogenase (17β-HSD, and 17α-hydroxylase were examined by Western blotting and semi-quantitative RT-PCR, respectively. Annexin A5 significantly stimulated testosterone secretion from rat Leydig cells in dose- and time-dependent manners and increased mRNA and protein expression of StAR, P450scc, 3β-HSD, and 17β-HSD but not 17α-hydroxylase. Annexin A5 knockdown by siRNA significantly decreased the level of testosterone and protein expression of P450scc, 3β-HSD, and 17β-HSD. The significant activation of ERK1/2 signaling was observed at 5, 10, and 30 min after annexin A5 treatment. After the pretreatment of Leydig cells with ERK inhibitor PD98059 (50 μmol l−1 for 20 min, the effects of annexin A5 on promoting testosterone secretion and increasing the expression of P450scc, 3β-HSD, and 17β-HSD were completely abrogated (P < 0.05. Thus, ERK1/2 signaling is involved in the roles of annexin A5 in mediating testosterone production and the expression of P450scc, 3β-HSD, and 17β-HSD in Leydig cells.

  10. Reduced basal and novelty-induced levels of activity-regulated cytoskeleton associated protein (Arc) and c-Fos mRNA in the cerebral cortex and hippocampus of APPswe/PS1ΔE9 transgenic mice

    DEFF Research Database (Denmark)

    Christensen, Ditte Z; Thomsen, Morten Skøtt; Mikkelsen, Jens D

    2013-01-01

    in APP/PS1ΔE9 compared to wild-type mice. Novelty exposure induced an increase in Arc and c-Fos mRNA in the medial prefrontal cortex (mPFC), parietal cortex, and hippocampal formation in both APP/PS1ΔE9 transgenic and wild-type mice. However, novelty-induced IEG expression did not reach the same levels...

  11. The Canadian Natural Health Products (NHP regulations: industry perceptions and compliance factors

    Directory of Open Access Journals (Sweden)

    Boon Heather

    2006-05-01

    Full Text Available Abstract Background The use of natural health products, such as vitamins, minerals, and herbs, by Canadians has been increasing with time. As a result of consumer concern about the quality of these products, the Canadian Department of Health created the Natural Health Products (NHP Regulations. The new Canadian regulations raise questions about whether and how the NHP industry will be able to comply and what impact they will have on market structure. The objectives of this study were to explore who in the interview sample is complying with Canada's new NHP Regulations (i.e., submitted product licensing applications on time; and explore the factors that affect regulatory compliance. Methods Twenty key informant interviews were conducted with employees of the NHP industry. The structured interviews focused on the level of satisfaction with the Regulations and perceptions of compliance and non-compliance. Interviews were tape recorded and then transcribed verbatim. Data were independently coded, using qualitative content analysis. Team meetings were held after every three to four interviews to discuss emerging themes. Results The major finding of this study is that most (17 out of 20 companies interviewed were beginning to comply with the new regulatory regime. The factors that contribute to likelihood of regulatory compliance were: perceptions and knowledge of the regulations and business size. Conclusion The Canadian case can be instructive for other countries seeking to implement regulatory standards for natural health products. An unintended consequence of the Canadian NHP regulations may be the exit of smaller firms, leading to industry consolidation.

  12. The ACAT inhibitor VULM1457 significantly reduced production and secretion of adrenomedullin (AM) and down-regulated AM receptors on human hepatoblastic cells.

    Science.gov (United States)

    Drímal, J; Fáberová, V; Schmidtová, L; Bednáriková, M; Drímal, J; Drímal, D

    2005-12-01

    Acyl-CoA:cholesterol acyltransferase (ACAT) is an important enzyme in the pathways of cholesterol esterification. It has been shown that new ACAT inhibitor 1-(2,6-diisopropyl-phenyl)-3-[4-(4'-nitrophenylthio)phenyl] urea (VULM1457) significantly reduced atherogenic activity in animal experimental atherosclerosis. Proliferative hormone adrenomedullin (AM) has been shown to be released in response to hypoxia, however, its role in cellular protection has remained elusive. The effect of increased local production of AM in cells and resultant down-regulation of AM receptors has not been investigated yet. We hypothesized that increased expression of AM in hypoxic cells was the result of excessive AM production with resultant AM receptor down-regulation, surface-membrane protein degradation and that the new specific ACAT inhibitor would reduce AM induction in hypoxia and thus proliferation of cells. In order to investigate specific cellular AM signaling and protection induced by VULM1457, we characterized specific surface-membrane [125I]AM receptors expressed on cells, evaluated AM secretion (RIA assays), AM mRNA expression in cultured cells (RT-PCR analysis) and proliferation (incorporation of [3H]thymidine) in control, hypoxic and metabolically stressed human hepatoblastoma cell lines exposed to gradually increasing concentrations of VULM1457. The new ACAT inhibitor VULM1457 in concentration 0.03 and 0.1 micromol/l significantly down-regulated specific AM receptors on HepG2 cells, reduced AM secretion of HepG2 cells exposed to hypoxia. These results suggest that VULM1457, as new member of ACAT family of inhibitors could negatively regulate cell proliferation induced by AM, which may correlate with down-regulation of membrane-bound AM receptors on HepG2 cells, and moreover, with the induction and expression of AM in hypoxia.

  13. A CRE1- regulated cluster is responsible for light dependent production of dihydrotrichotetronin in Trichoderma reesei.

    Directory of Open Access Journals (Sweden)

    Alberto Alonso Monroy

    Full Text Available Changing light conditions, caused by the rotation of earth resulting in day and night or growth on the surface or within a substrate, result in considerably altered physiological processes in fungi. For the biotechnological workhorse Trichoderma reesei, regulation of glycoside hydrolase gene expression, especially cellulase expression was shown to be a target of light dependent gene regulation. Analysis of regulatory targets of the carbon catabolite repressor CRE1 under cellulase inducing conditions revealed a secondary metabolite cluster to be differentially regulated in light and darkness and by photoreceptors. We found that this cluster is involved in production of trichodimerol and that the two polyketide synthases of the cluster are essential for biosynthesis of dihydrotrichotetronine (syn. bislongiquinolide or bisorbibutenolide. Additionally, an indirect influence on production of the peptaibol antibiotic paracelsin was observed. The two polyketide synthetase genes as well as the monooxygenase gene of the cluster were found to be connected at the level of transcription in a positive feedback cycle in darkness, but negative feedback in light, indicating a cellular sensing and response mechanism for the products of these enzymes. The transcription factor TR_102497/YPR2 residing within the cluster regulates the cluster genes in a light dependent manner. Additionally, an interrelationship of this cluster with regulation of cellulase gene expression was detected. Hence the regulatory connection between primary and secondary metabolism appears more widespread than previously assumed, indicating a sophisticated distribution of resources either to degradation of substrate (feed or to antagonism of competitors (fight, which is influenced by light.

  14. Postgraduate education and research in Brazil: regulation and reconfiguration processes of academic work formation and production

    Directory of Open Access Journals (Sweden)

    João Ferreira de Oliveira

    2015-07-01

    Full Text Available This text analyses some of the processes of formation and production regulation and reconfiguration of the scholarly work in Brazil. Initially we examine the context and meaning of knowledge production in times of flexible accumulation, as well as the current landscape of Postgraduate education in the country. We seek to understand how public policies in the area, particularly the actions of evaluation and promotion, and the new modus operandi of the Postgraduate study and research organization have been reconfiguring the work production of teaching and students within the programs, especially in education. Above all, we seek to highlight the role of promotion and evaluation agencies, increasingly committed to a vision of expansion that drives the production of knowledge associated with demands of economic-productivity, rather than a consistent formative project that would result in a significant advancement in the production and dissemination of knowledge in the different areas.

  15. Leptin deficiency down-regulates IL-23 production in glomerular podocytes resulting in an attenuated immune response in nephrotoxic serum nephritis.

    Science.gov (United States)

    Goto, Kei; Kaneko, Yoshikatsu; Sato, Yuya; Otsuka, Tadashi; Yamamoto, Suguru; Goto, Shin; Yamamoto, Keiko; Yamamoto, Tadashi; Kawachi, Hiroshi; Madaio, Michael P; Narita, Ichiei

    2016-04-01

    Leptin, one of the typical adipokines, is reported to promote Th17 cell responses and to enhance production of proinflammatory cytokines. To clarify the role of leptin in the regulation of the IL-23/IL-17 axis and the development of kidney disease, we used a murine model of nephrotoxic serum (NTS) nephritis (NTN). Sheep NTS was administered in wild-type C57BL/6J mice and food-restricted, leptin-deficient C57BL/6J-ob/ob(FR-ob/ob) mice after preimmunization with sheep IgG. The profile of mRNA expression relevant to T helper lymphocytes in the kidneys was analyzed by quantitative real-time PCR (qRT-PCR). Cultured murine glomerular podocytes and peritoneal exudate macrophages (PEMs) were used to investigate the direct effect of leptin on IL-23 or MCP-1 production by qRT-PCR. Kidney injury and macrophage infiltration were significantly attenuated in FR-ob/obmice 7 days after NTS injection. The Th17-dependent secondary immune response against deposited NTS in the glomeruli was totally impaired in FR-ob/obmice because of deteriorated IL-17 and proinflammatory cytokine production including IL-23 and MCP-1 in the kidney. IL-23 was produced in glomerular podocytes in NTN mice and cultured murine glomerular podocytes produced IL-23 under leptin stimulation. MCP-1 production in PEMs was also promoted by leptin. Induction of MCP-1 expression was observed in PEMs regardless of Ob-Rb, and the leptin signal was transduced without STAT3 phosphorylation in PEMs. Leptin deficiency impairs the secondary immune response against NTS and down-regulates IL-23 production and Th17 responses in the NTN kidney, which is accompanied by decreased MCP-1 production and macrophage infiltration in the NTN kidney. © The Japanese Society for Immunology. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Leptin deficiency down-regulates IL-23 production in glomerular podocytes resulting in an attenuated immune response in nephrotoxic serum nephritis

    Science.gov (United States)

    Goto, Kei; Sato, Yuya; Otsuka, Tadashi; Yamamoto, Suguru; Goto, Shin; Yamamoto, Keiko; Yamamoto, Tadashi; Kawachi, Hiroshi; Madaio, Michael P.; Narita, Ichiei

    2016-01-01

    Leptin, one of the typical adipokines, is reported to promote Th17 cell responses and to enhance production of proinflammatory cytokines. To clarify the role of leptin in the regulation of the IL-23/IL-17 axis and the development of kidney disease, we used a murine model of nephrotoxic serum (NTS) nephritis (NTN). Sheep NTS was administered in wild-type C57BL/6J mice and food-restricted, leptin-deficient C57BL/6J-ob/ob (FR-ob/ob) mice after preimmunization with sheep IgG. The profile of mRNA expression relevant to T helper lymphocytes in the kidneys was analyzed by quantitative real-time PCR (qRT–PCR). Cultured murine glomerular podocytes and peritoneal exudate macrophages (PEMs) were used to investigate the direct effect of leptin on IL-23 or MCP-1 production by qRT–PCR. Kidney injury and macrophage infiltration were significantly attenuated in FR-ob/ob mice 7 days after NTS injection. The Th17-dependent secondary immune response against deposited NTS in the glomeruli was totally impaired in FR-ob/ob mice because of deteriorated IL-17 and proinflammatory cytokine production including IL-23 and MCP-1 in the kidney. IL-23 was produced in glomerular podocytes in NTN mice and cultured murine glomerular podocytes produced IL-23 under leptin stimulation. MCP-1 production in PEMs was also promoted by leptin. Induction of MCP-1 expression was observed in PEMs regardless of Ob-Rb, and the leptin signal was transduced without STAT3 phosphorylation in PEMs. Leptin deficiency impairs the secondary immune response against NTS and down-regulates IL-23 production and Th17 responses in the NTN kidney, which is accompanied by decreased MCP-1 production and macrophage infiltration in the NTN kidney. PMID:26567290

  17. Tools Related to the Federal Tobacco Products Regulations: What Retailers Need to Know PSA (:30)

    Centers for Disease Control (CDC) Podcasts

    2010-09-16

    PSA to announce a new mobile text message program that will help raise retailers' awareness of the new federal tobacco regulations.  Created: 9/16/2010 by The CDC Division of News and Electronic Media and the FDA Center for Tobacco Products.   Date Released: 9/16/2010.

  18. Regulating Tobacco Product Advertising and Promotions in the Retail Environment: A Roadmap for States and Localities.

    Science.gov (United States)

    Lange, Tamara; Hoefges, Michael; Ribisl, Kurt M

    2015-01-01

    Recent amendments to federal law and a burgeoning body of research have intensified public health officials' interest in reducing youth initiation of tobacco use, including by regulating the time, place, or manner of tobacco product advertising at the point of sale. This article analyzes legal obstacles to various strategies for reducing youth initiation. © 2015 American Society of Law, Medicine & Ethics, Inc.

  19. One carbon metabolism in anaerobic bacteria: Regulation of carbon and electron flow during organic acid production

    Energy Technology Data Exchange (ETDEWEB)

    Zeikus, J.G.; Jain, M.K.

    1992-01-01

    This reporting period, progress is reported on the following: metabolic pathway of solvent production in B. methylotrophicum; the biochemical mechanism for metabolic regulation of the succinate fermentation; models to understand the physiobiochemical function of formate metabolism in anaerobes and; models for understanding the influence of low pH on one carbon metabolism. (CBS)

  20. Regulation of acidity and reduction of turbidity in the clarified pomegranate juice production

    OpenAIRE

    ESHMATOV FOZIL KHIDIROVICH; MAKSUMOVA DILRABO KUCHKAROVNA; DODAEVA LAYLO KUCHKAROVNA

    2016-01-01

    Regulation of acidity and reduction of turbidity in the clarified pomegranate juice production. From sour varieties of pomegranates may obtain normal natural pomegranate juice by anion-exchange resin. There are determined problems quantity of precipitate and unstable color in the pomegranate juice and concentrate by experimentally.

  1. Residual efficacy of the insect growth regulator pyriproxyfen for control of stored product insects

    Science.gov (United States)

    The insect growth regulator pyriproxyfen is registered in the USA as an aerosol and as a surface treatment to control stored product insects. Field trials with the aerosol show that residues from an application of pyrethrin + pyriproxyfen gave residual control of the red flour beetle, Tribolium cast...

  2. Heat-killed probiotic bacteria differentially regulate colonic epithelial cell production of human β-defensin-2: dependence on inflammatory cytokines.

    Science.gov (United States)

    Habil, N; Abate, W; Beal, J; Foey, A D

    2014-12-01

    The inducible antimicrobial peptide human β-defensin-2 (hBD-2) stimulated by pro-inflammatory cytokines and bacterial products is essential to antipathogen responses of gut epithelial cells. Commensal and probiotic bacteria can augment such mucosal defences. Probiotic use in the treatment of inflammatory bowel disease, however, may have adverse effects, boosting inflammatory responses. The aim of this investigation was to determine the effect of selected probiotic strains on hBD-2 production by epithelial cells induced by pathologically relevant pro-inflammatory cytokines and the role of cytokine modulators in controlling hBD-2. Caco-2 colonic intestinal epithelial cells were pre-incubated with heat-killed probiotics, i.e. Lactobacillus casei strain Shirota (LcS) or Lactobacillus fermentum strain MS15 (LF), followed by stimulation of hBD-2 by interleukin (IL)-1β and tumour necrosis factor alpha (TNF-α) in the absence or presence of exogenous IL-10 or anti-IL-10 neutralising antibody. Cytokines and hBD-2 mRNA and protein were analysed by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. LcS augmented IL-1β-induced hBD-2, whereas LF enhanced TNF-α- and suppressed IL-1β-induced hBD-2. LF enhanced TNF-α-induced TNF-α and suppressed IL-10, whereas augmented IL-1β-induced IL-10. LcS upregulated IL-1β-induced TNF-α mRNA and suppressed IL-10. Endogenous IL-10 differentially regulated hBD-2; neutralisation of IL-10 augmented TNF-α- and suppressed IL-1β-induced hBD-2. Exogenous IL-10, however, suppressed both TNF-α- and IL-1β-induced hBD-2; LcS partially rescued suppression in TNF-α- and IL-1β-stimulation, whereas LF further suppressed IL-1β-induced hBD-2. It can be concluded that probiotic strains differentially regulate hBD-2 mRNA expression and protein secretion, modulation being dictated by inflammatory stimulus and resulting cytokine environment.

  3. Glutamate affects the production of epoxyeicosanoids within the brain: The up-regulation of brain CYP2J through the MAPK-CREB signaling pathway.

    Science.gov (United States)

    Liu, Mingzhou; Zhu, Quanfei; Wu, Jinhua; Yu, Xuming; Hu, Mingbai; Xie, Xianfei; Yang, Zheqiong; Yang, Jing; Feng, Yu-Qi; Yue, Jiang

    2017-04-15

    Glutamate is the major excitatory neurotransmitter in the brain, and chronic glutamate excitotoxicity has been thought to be involved in numerous neurodegenerative diseases. We investigated the effects of glutamate at concentrations lower than the usual extrasynaptic concentrations on the production of epoxyeicosanoids mediated by brain CYP2J. Glutamate increased CYP2J2 mRNA levels in astrocytes in a dose-dependent manner, while an antagonist of the metabotropic glutamate receptor subtype 5 (mGlu5 receptor) attenuated the glutamate-induced increases in CYP2J2 levels by glutamate. Glutamate increased the binding of cAMP response element-binding protein (CREB) with the CYP2J2 promoter, and the inhibition of the MAPK signaling pathway (ERK1/2, p38, and JNK) decreased the binding of CREB with the CYP2J2 promoter following the glutamate treatment. CREB activated the CYP2J2 promoter located at -1522 to -1317bp, and CREB overexpression significantly increased CYP2J2 mRNA levels. The CYP2J2 and mGlu5 mRNA levels were higher in the frontal cortex, hippocampus, cerebellum, and brainstem in adult rats that received a subcutaneous injection of monosodium l-glutamate at 1, 3, 5, and 7days of age. The data from the partial least-squares-discriminant analysis showed the epoxyeicosanoid profile of the hippocampus from the cerebellum, brain stem, and frontal cortex. The sum of the epoxyeicosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids (DHETs) was increased by 1.16-fold, 1.18-fold, and 1.19-fold in the frontal cortex, cerebellum, and brain stem, respectively, in rats treated with monosodium l-glutamate compared with the control group. The results suggest that brain CYP2J levels and CYP2J-mediated epoxyeicosanoid production can be regulated by extrasynaptic glutamate. The glutamate receptors expressed in astrocytes may mediate the regulation of drug-metabolizing enzymes and the metabolome of endogenous substances by glutamate. Copyright © 2017 Elsevier B.V. All rights

  4. Glucagon-Like Peptide-1 Secreting Cell Function as well as Production of Inflammatory Reactive Oxygen Species Is Differently Regulated by Glycated Serum and High Levels of Glucose

    Directory of Open Access Journals (Sweden)

    Alessandra Puddu

    2014-01-01

    Full Text Available Glucagon-like peptide-1 (GLP-1, an intestinal hormone contributing to glucose homeostasis, is synthesized by proglucagon and secreted from intestinal neuroendocrine cells in response to nutrients. GLP-1 secretion is impaired in type 2 diabetes patients. Here, we aimed at investigating whether diabetic toxic products (glycated serum (GS or high levels of glucose (HG may affect viability, function, and insulin sensitivity of the GLP-1 secreting cell line GLUTag. Cells were cultured for 5 days in presence or absence of different dilutions of GS or HG. GS and HG (alone or in combination increased reactive oxygen species (ROS production and upregulated proglucagon mRNA expression as compared to control medium. Only HG increased total production and release of active GLP-1, while GS alone abrogated secretion of active GLP-1. HG-mediated effects were associated with the increased cell content of the prohormone convertase 1/3 (PC 1/3, while GS alone downregulated this enzyme. HG upregulated Glucokinase (GK and downregulated SYNTHAXIN-1. GS abrogated SYNTHAXIN-1 and SNAP-25. Finally, high doses of GS alone or in combination with HG reduced insulin-mediated IRS-1 phosphorylation. In conclusion, we showed that GS and HG might regulate different pathways of GLP-1 production in diabetes, directly altering the function of neuroendocrine cells secreting this hormone.

  5. Sensitivity of grassland productivity to aridity controlled by stomatal and xylem regulation

    Science.gov (United States)

    Konings, A. G.; Williams, A. P.; Gentine, P.

    2017-03-01

    The terrestrial water and carbon cycles are coupled through plant regulation of stomatal closure. Both soil moisture and vapour pressure deficit--the amount of moisture in the air relative to its potential maximum--can govern stomatal closure, which reduces plant carbon uptake. However, plants vary in the degree to which they regulate their stomata--and in association, xylem conductance--in response to increasing aridity: isohydric plants exert tight regulation of stomata and the water content of the plant, whereas anisohydric plants do not. Here we use remote-sensing data sets of anisohydricity and vegetation greenness to show that productivity in United States grasslands--especially anisohydric ones--is far more sensitive to variations in vapour pressure deficit than to variations in precipitation. Anisohydric ecosystem productivity is over three times more sensitive to vapour pressure deficit than isohydric ecosystem productivity. The precipitation sensitivity of summer productivity increases with anisohydricity only for the most anisohydric ecosystems. We conclude that increases in vapour pressure deficit rather than changes in precipitation--both of which are expected impacts of climate change--will be a dominant influence on future grassland productivity.

  6. Electron acceptor-based regulation of microbial greenhouse gas production from thawing permafrost

    DEFF Research Database (Denmark)

    Bak, Ebbe Norskov; Jones, Eleanor; Yde, Jacob Clement

    of sulfate and iron and the microbial community structure regulate the production of CO2 and CH4 in thawing permafrost, and to elucidate how the rate of the organic carbon degradation changes with depth in permafrost-affected soils. This study improves our understanding of climate feedback mechanisms......Permafrost contains about 35% of the global soil organic carbon (0-3 m depth). As a consequence of global warming, the active layer thickness is steadily increasing and its organic carbon is becoming available for degradation, causing a concomitant release of CO2 and CH4. The climate forcing...... feedbacks of permafrost thaw are determined by the rate of organic carbon degradation and to which degree it is released as CO2 or CH4. Methane is produced under anoxic conditions, but the factors that regulate its production are poorly constrained. In this study, we investigate how CH4 production...

  7. [Effects of menthol as an additive in tobacco products and the need for regulation].

    Science.gov (United States)

    Kahnert, S; Nair, U; Mons, U; Pötschke-Langer, M

    2012-03-01

    Menthol is the most widely used and the most prominent tobacco additive in tobacco products advertised and marketed by the tobacco industry. Besides its characteristic flavor, it possesses a variety of pharmacological properties facilitating tobacco smoke inhalation and potentiating dependence. These properties of menthol not only favor tobacco initiation and consumption but can also prevent smoking cessation. This article summarizes the effect of menthol as an additive in tobacco products and its effect on tobacco consumption that causes a number of chronic diseases and premature death and, therefore, counteracts tobacco control measures. Currently, there is no legislative regulation in Germany that considers the health hazard, addiction-enhancing and attractiveness-increasing properties of additives permitted in tobacco products. Effective regulation or even a ban could contribute to a reduction of tobacco consumption and, hence, save many people from a long-lasting tobacco dependence.

  8. Biotechnological production and application of the antibiotic pimaricin: biosynthesis and its regulation.

    Science.gov (United States)

    Aparicio, Jesús F; Barreales, Eva G; Payero, Tamara D; Vicente, Cláudia M; de Pedro, Antonio; Santos-Aberturas, Javier

    2016-01-01

    Pimaricin (natamycin) is a small polyene macrolide antibiotic used worldwide. This efficient antimycotic and antiprotozoal agent, produced by several soil bacterial species of the genus Streptomyces, has found application in human therapy, in the food and beverage industries and as pesticide. It displays a broad spectrum of activity, targeting ergosterol but bearing a particular mode of action different to other polyene macrolides. The biosynthesis of this only antifungal agent with a GRAS status has been thoroughly studied, which has permitted the manipulation of producers to engineer the biosynthetic gene clusters in order to generate several analogues. Regulation of its production has been largely unveiled, constituting a model for other polyenes and setting the leads for optimizing the production of these valuable compounds. This review describes and discusses the molecular genetics, uses, mode of action, analogue generation, regulation and strategies for increasing pimaricin production yields.

  9. Attenuation in the rph-pyrE operon of Escherichia coli and processing of the dicistronic mRNA

    DEFF Research Database (Denmark)

    Poulsen, Peter; Jensen, Kaj Frank

    1992-01-01

    , the dicistronic rph-pyrE transcript was rapidly processed into two monocistronic mRNA units, and a cleavage site was mapped near the attenuator in the intercistronic region, close to the site where transcription was terminated in high-UTP pools. Furthermore, the major 3' end of the pyrE mRNA was mapped near......We have substituted on a plasmid the native promoter of the Escherichia coli rph-pyrE operon with an inducible transcription-initiation signal. The plasmid was used to study the mRNA chains derived from the operon at different intracellular concentrations of UTP and as a function of time following...... induction of transcription. The results showed that dicistronic rph-pyrE mRNA was formed when the UTP pool was low, and that a monocistronic rph mRNA was the major transcription product in high-UTP pools, thus supporting an UTP-controlled attenuation mechanism for regulation of pyrE gene expression. However...

  10. Engineering of an Lrp family regulator SACE_Lrp improves erythromycin production in Saccharopolyspora erythraea.

    Science.gov (United States)

    Liu, Jing; Chen, Yunfu; Wang, Weiwei; Ren, Min; Wu, Panpan; Wang, Yansheng; Li, Changrun; Zhang, Lixin; Wu, Hang; Weaver, David T; Zhang, Buchang

    2017-01-01

    Leucine-responsive regulatory proteins (Lrps) are a group of transcriptional regulators that regulate diverse cellular processes in bacteria and archaea. However, the regulatory role of Lrps in antibiotic biosynthesis remains poorly understood. In this study, we show that SACE_5388, an Lrp family regulator named as SACE_Lrp, is an efficient regulator for transporting and catabolizing branched-chain amino acids (BCAAs), playing an important role in regulating erythromycin production in Saccharopolyspora erythraea. SACE_Lrp directly controlled the expression of the divergently transcribed SACE_5387-5386 operon putatively encoding a BCAA ABC transporter by interacting with the intergenic region between SACE_Lrp and SACE_5387 (SACE_Lrp-5387-int), and indirectly controlled the expression of ilvE putatively encoding an aminotransferase catabolizing BCAAs. BCAA catabolism is one source of the precursors for erythromycin biosynthesis. Lysine and arginine promoted the dissociation of SACE_Lrp from SACE_Lrp -5387-int, whereas histidine increased their binding. Gene disruption of SACE_Lrp (ΔSACE_Lrp) in S. erythraea A226 resulted in a 25% increase in erythromycin production, while overexpression of SACE_5387-5386 in A226 enhanced erythromycin production by 36%. Deletion of SACE_Lrp (WBΔSACE_Lrp) in the industrial strain S. erythraea WB enhanced erythromycin production by 19%, and overexpression of SACE_5387-5386 in WBΔSACE_Lrp (WBΔSACE_Lrp/5387-5386) increased erythromycin production by 41% compared to WB. Additionally, supplement of 10mM valine to WBΔSACE_Lrp/5387-5386 culture further increased total erythromycin production up to 48%. In a 5-L fermenter, the erythromycin accumulation in the engineered strain WBΔSACE_Lrp/5387-5386 with 10mM extra valine in the industrial culture media reached 5001mg/L, a 41% increase over 3503mg/L of WB. These insights into the molecular regulation of antibiotic biosynthesis by SACE_Lrp in S. erythraea are instrumental in increasing

  11. Performance-based regulation: enterprise responsibility for reducing death, injury, and disease caused by consumer products.

    Science.gov (United States)

    Sugarman, Stephen D

    2009-12-01

    This article offers a bold new idea for confronting the staggering level of death, injury, and disease caused by five consumer products: cigarettes, alcohol, guns, junk food, and motor vehicles. Business leaders try to frame these negative outcomes as "collateral damage" that is someone else's problem. That framing not only is morally objectionable but also overlooks the possibility that, with proper prodding, industry could substantially lessen these public health disasters. I seek to reframe the public perception of who is responsible and propose to deploy a promising approach called "performance-based regulation" to combat the problem. Performance-based regulation would impose on manufacturers a legal obligation to reduce the negative social costs of their products. Rather than involving them in litigation or forcing them to operate differently (as "command-and-control" regimes do), performance-based regulation allows the firms to determine how best to decrease bad public health consequences. Like other public health strategies, performance-based regulation focuses on those who are far more likely than individual consumers to achieve real gains. Analogous to a tax on causing harm that exceeds a threshold level, performance-based regulation seeks to harness private initiative in pursuit of the public good.

  12. Heme metabolism in stress regulation and protein production: From Cinderella to a key player.

    Science.gov (United States)

    Martínez, J L; Petranovic, D; Nielsen, J

    2016-04-02

    Heme biosynthesis is a highly conserved pathway which is present in all kingdoms, from Archaea to higher organisms such as plants and mammals. The heme molecule acts as a prosthetic group for different proteins and enzymes involved in energy metabolism and reactions involved in electron transfer. Based on our recent findings and other recent reports, we here illustrate that heme is more than a co-factor. We also discuss the necessity to gain more insight into the heme biosynthesis pathway regulation, as this interacts closely with overall stress control. Understanding heme biosynthesis and its regulation could impact our ability to develop more efficient yeast cell factories for heterologous protein production.

  13. MicroRNA-155 Regulates ROS Production, NO Generation, Apoptosis and Multiple Functions of Human Brain Microvessel Endothelial Cells Under Physiological and Pathological Conditions.

    Science.gov (United States)

    Liu, Yajing; Pan, Qunwen; Zhao, Yuhui; He, Caixia; Bi, Kexia; Chen, Yusen; Zhao, Bin; Chen, Yanfang; Ma, Xiaotang

    2015-12-01

    The microRNA-155 (miR155) regulates various functions of cells. Dysfunction or injury of endothelial cells (ECs) plays an important role in the pathogenesis of various vascular diseases. In this study, we investigated the role and potential mechanisms of miR155 in human brain microvessel endothelial cells (HBMECs) under physiological and pathological conditions. We detected the effects of miR155 silencing on ROS production, NO generation, apoptosis and functions of HBMECs at basal and in response to oxidized low density lipoprotein (ox-LDL). Western blot and q-PCR were used for analyzing the gene expression of epidermal growth factor receptor (EGFR)/extracellular regulated protein kinases (ERK)/p38 mitogen-activated protein kinase (p38 MAPK), phosphatidylinositol-3-kinase (PI3K) and serine/threonine kinase(Akt), activated caspase-3, and intercellular adhesion molecule-1 (ICAM-1). Results showed that under both basal and challenge situations: (1) Silencing of miR155 decreased apoptosis and reactive oxygen species (ROS) production of HBMECs, whereas, promoted nitric oxide (NO) generation. (2) Silencing of miR155 increased the proliferation, migration, and tube formation ability of HBMECs, while decreased cell adhesion ability. (3) Gene expression analyses showed that EGFR/ERK/p38 MAPK and PI3K/Akt were increased and that activated caspase-3 and ICAM-1 mRNA were decreased after knockdown of miR155. In conclusion, knockdown of miR155 could modulate ROS production, NO generation, apoptosis and function of HBMECs via regulating diverse gene expression, such as caspase-3, ICAM-1 and EGFR/ERK/p38 MAPK and PI3K/Akt pathways. © 2015 Wiley Periodicals, Inc.

  14. A mathematical model of quorum sensing regulated EPS production in biofilm communities

    Science.gov (United States)

    2011-01-01

    Background Biofilms are microbial communities encased in a layer of extracellular polymeric substances (EPS). The EPS matrix provides several functional purposes for the biofilm, such as protecting bacteria from environmental stresses, and providing mechanical stability. Quorum sensing is a cell-cell communication mechanism used by several bacterial taxa to coordinate gene expression and behaviour in groups, based on population densities. Model We mathematically model quorum sensing and EPS production in a growing biofilm under various environmental conditions, to study how a developing biofilm impacts quorum sensing, and conversely, how a biofilm is affected by quorum sensing-regulated EPS production. We investigate circumstances when using quorum-sensing regulated EPS production is a beneficial strategy for biofilm cells. Results We find that biofilms that use quorum sensing to induce increased EPS production do not obtain the high cell populations of low-EPS producers, but can rapidly increase their volume to parallel high-EPS producers. Quorum sensing-induced EPS production allows a biofilm to switch behaviours, from a colonization mode (with an optimized growth rate), to a protection mode. Conclusions A biofilm will benefit from using quorum sensing-induced EPS production if bacteria cells have the objective of acquiring a thick, protective layer of EPS, or if they wish to clog their environment with biomass as a means of securing nutrient supply and outcompeting other colonies in the channel, of their own or a different species. PMID:21477365

  15. The plant growth regulator methyl jasmonate inhibits aflatoxin production by Aspergillus flavus.

    Science.gov (United States)

    Goodrich-Tanrikulu, M; Mahoney, N E; Rodriguez, S B

    1995-11-01

    Aflatoxins are highly toxic and carcinogenic compounds produced by certain Aspergillus species on agricultural commodities. The presence of fatty acid hydroperoxides, which can form in plant material either preharvest under stress or postharvest under improper storage conditions, correlates with high levels of aflatoxin production. Effects on fungal growth and aflatoxin production are known for only a few of the numerous plant metabolites of fatty acid hydroperoxides. Jasmonic acid (JA), a plant growth regulator, is a metabolite of 13-hydroperoxylinolenic acid, derived from alpha-linolenic acid. The volatile methyl ester of JA, methyl jasmonate (MeJA), is also a plant growth regulator. In this study we report the effect of MeJA on aflatoxin production and growth of Aspergillus flavus. MeJA at concentrations of 10(-3)-10(-8) M in the growth medium inhibited aflatoxin production, by as much as 96%. Exposure of cultures to MeJA vapour similarly inhibited aflatoxin production. The amount of aflatoxin produced depended on the timing of the exposure. MeJA treatment also delayed spore germination and inhibited the production of a mycelial pigment. These fungal responses resemble plant jasmonate responses.

  16. Epigenetic Regulation of Cytokine Production in Human Amnion and Villous Placenta

    Directory of Open Access Journals (Sweden)

    Murray D. Mitchell

    2012-01-01

    Full Text Available The mechanisms of human preterm labour appear inextricably linked to cytokine biosynthesis by gestational tissues. In turn, cytokine production by gestational tissues has been shown to be regulated by epigenetic mechanisms. In this paper, we demonstrate that cytokine production in gestational tissues is regulated epigenetically in a tissue-specific manner. Furthermore, we show that treatment with a histone deacetylation inhibitor can partially abrogate LPS-stimulated TNFα production in villous placenta but not amnion. LPS treatment significantly (100-fold and IL10 (~6–10-fold after 24 h of treatment in villous explants, as expected. There were no significant LPS effects on IL1Ra production. AZA treatment did not have any significant effect on any cytokines' production tested either alone or in combination with LPS. Interestingly, however, the stimulatory effects of LPS on TNFα production were partially mitigated (<0.05 by TSA treatment in villous explants. We suggest caution in the consideration of histone deacetylation inhibitors in pregnancy due to the different responses in gestational tissues.

  17. FEATURES OF THE STATE REGULATION OF THE PRODUCTION OF GENETICALLY MODIFIED PRODUCTS IN THE WORLD AND IN UKRAINE

    Directory of Open Access Journals (Sweden)

    Viktoriya Bashuk

    2017-11-01

    Full Text Available The purpose of this article is to investigate the regulation of the use, consumption, and trade of genetically modified organisms in different countries of the world, as well as in Ukraine. The definition of international approaches to risk assessment of genetically modified products is of particular importance for international trade. Methodology. The study is based on data from different sources, beginning with the first mention of genetically modified organisms, ending with the latest received data from different countries. Purpose. Show how different countries refer differently to the production of genetically modified products, differently perceive it and are guided by different principles. Find ways to solve the problems associated with the introduction of GMOs in Ukraine and compare them with other countries. Results. The study showed that developed countries have developed clear rules for the production, labelling, consumption, and trade of products containing GMOs. Also, the bodies and structures responsible for compliance with all these rules are defined and a large number of legislative acts has been adopted, which cannot be said of Ukraine. In Ukraine, this is a large gap because “on paper” also seems that there are some rules according to GMOs but they are not clear, consistent, and they are not followed due to their observance, as these powers are entrusted to a large number of structures. Due to imperfect legislation and lack of funds, products that are imported are not tested for GMOs content, there are no studies on the safety of their consumption and cultivation, the reliability of information on labels is not followed. Practical implications. In Ukraine, in order to ensure the proper level of state regulation, protection, and use of genetically modified products obtained with the help of modern biotechnologies, it is necessary to adhere strictly to the fulfilment of the main criteria: 1 adoption and further improvement of

  18. Production of pigments in Alternanthera sessilis calli mediated by plant growth regulators and light

    Directory of Open Access Journals (Sweden)

    Cristini Milech

    2017-09-01

    Full Text Available Among the compounds produced by plants, pigments such as betalains have received attention from both food and pharmaceuticals industries. The Alternanthera sessilis species produces these pigments, though in small quantities, and so it is necessary to increase production. Thus, many studies use elicitors that are capable of triggering physiological or morphological responses in plants. The objective was to establish callus production in A. sessilis grown under different combinations of growth regulators and light qualities and to assess whether these factors can increase betalain and flavonoid production. Leaf and internodal explants in MS medium with different growth regulators were used to obtain calli, which were subsequently transferred to a betacyanin induction medium remaining for 40 days under different light qualities (white, blue, red, and dark. The most suitable treatment for callus formation and subsequent betalain and flavonoid induction was to combine a medium containing 6.7 µmol L-1 2,4-D and 9.0 µmol L-1 BAP and blue light. Physical elicitation by light combined with appropriate concentration of growth regulators on calli can increase production of commercially important metabolites.

  19. Navigating through orphan medicinal product regulations in EU and US--similarities and differences.

    Science.gov (United States)

    Tiwari, Jyoti

    2015-02-01

    Rare diseases as the name suggests are the diseases which occur in a very small population due to which the development of medicinal products for these diseases is sidelined as it is anticipated that the cost of development will never be recovered from the sales. It has been estimated by National Institute of Health (NIH) that globally around 7000 rare diseases are there, many of which are of genetic origin. This paper aims to analyze the basic similarities and differences between the rules and regulations put forth by regulatory agencies of US and EU for development of medicinal products for rare diseases, also called orphan medicinal products. The basic purpose was to carve out the loopholes as well as positive aspects of each of these acts and regulations so as to have a clear understanding on the subject. It was to understand that how these legal instruments have stimulated the growth of the drug products for rare diseases and what other things can be done in order to achieve a better impact. This article also provides an overview of the various incentives offered as well as challenges and hurdles faced by each of these regulatory agencies while implementing these regulations. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Regulation of Cell Component Production by Growth Rate in the Group B Streptococcus

    Science.gov (United States)

    Ross, Robin A.; Madoff, Lawrence C.; Paoletti, Lawrence C.

    1999-01-01

    Group B Streptococcus (GBS) is the leading cause of bacterial sepsis and meningitis among neonates. While the capsular polysaccharide (CPS) is an important virulence factor of GBS, other cell surface components, such as C proteins, may also play a role in GBS disease. CPS production by GBS type III strain M781 was greater when cells were held at a fast (1.4-h mass-doubling time [td]) than at a slow (11-h td) rate of growth. To further investigate growth rate regulation of CPS production and to investigate production of other cell components, different serotypes and strains of GBS were grown in continuous culture in a semidefined and a complex medium. Samples were obtained after at least five generations at the selected growth rate. Cells and cell-free supernatants were processed immediately, and results from all assays were normalized for cell dry weight. All serotypes (Ia, Ib, and III) and strains (one or two strains per serotype) tested produced at least 3.6-fold more CPS at a td of 1.4 h than at a td of 11 h. Production of beta C protein by GBS type Ia strain A909 and type Ib strain H36B was also shown to increase at least 5.5-fold with increased growth rate (production at a td of 1.4 h versus 11 h). The production of alpha C protein by the same strains did not significantly change with increased growth rate. The effect of growth rate on other cell components was also investigated. Production of group B antigen did not change with growth rate, while alkaline phosphatase decreased with increased growth rate. Both CAMP factor and beta-hemolysin production increased fourfold with increased growth rate. Growth rate regulation is specific for select cell components in GBS, including beta C protein, alkaline phosphatase, beta-hemolysin, and CPS production. PMID:10464211

  1. 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

    The kinetics of mRNA expression in the central nervous system (CNS) for a series of putatively disease-promoting and disease-limiting cytokines during the course of experimental autoimmune encephalomyelitis (EAE) in Lewis rats were studied. Cytokine mRNA-expressing cells were detected...... proliferation and activation of T helper 1 (Th1) type cells producing IFN-gamma. The TNF-beta mRNA expression prior to onset of clinical signs favours a role for this cytokine in disease initiation. A pathogenic effector role of TNF-alpha was suggested from these observations that TNF-alpha mRNA expression...... of putative disease-promoting and -limiting cytokines in the CNS during acute monophasic EAE....

  2. Reproductive toxicity of a mixture of regulated drinking-water disinfection by-products in a multigenerational rat bioassay

    Science.gov (United States)

    BACKGROUND:Trihalomethanes (THMs) and haloaretic acids (HAAs) are regulated disinfection by-products (DBPs); their joint reproductive toxicity in drinking water is unknown.OBJECTIVE: We aimed to evaluate a drinking water mixture of the four regulated THMs and five regulated HAAs ...

  3. MiR-506 suppresses liver cancer angiogenesis through targeting sphingosine kinase 1 (SPHK1) mRNA.

    Science.gov (United States)

    Lu, Zhanping; Zhang, Weiying; Gao, Shan; Jiang, Qiulei; Xiao, Zelin; Ye, Lihong; Zhang, Xiaodong

    MicroRNAs acting as oncogenes or tumor suppressor genes play crucial roles in human cancers. Sphingosine kinase 1 (SPHK1) and its metabolite sphingosine 1-phosphate (S1P) contribute to tumor angiogenesis. We have reported that the down-regulation of miR-506 targeting YAP mRNA results in the hepatocarcinogenesis. In the present study, we report a novel function of miR-506, which suppresses tumor angiogenesis through targeting SPHK1 mRNA in liver cancer. Bioinformatics analysis showed that miR-506 might target 3'-untranslated region (3'UTR) of SPHK1 mRNA. Then, we validated that by luciferase reporter gene assays. MiR-506 was able to reduce the expression of SPHK1 at the levels of mRNA and protein using reverse transcription-polymerase chain reaction and Western blot analysis in hepatoma HepG2 cells. Functionally, human umbilical vein endothelial cell (HUVEC) tube formation assays demonstrated that the forced miR-506 expression remarkably inhibited the production of S1P in the supernatant of hepatoma cells. The supernatant resulted in the inhibition of tumor angiogenesis. Interestingly, the supernatant with overexpression of SPHK1 could rescue the inhibition of angiogenesis of liver cancer mediated by miR-506. Anti-miR-506 increased the production of S1P in the supernatant of hepatoma cells, but the supernatant with silencing of SPHK1 abolished anti-miR-506-induced acceleration of tumor angiogenesis. Clinically, we observed that the levels of miR-506 were negatively related to those of SPHK1 mRNA in liver cancer tissues. Thus, we conclude that miR-506 depresses the angiogenesis of liver cancer through targeting 3'UTR of SPHK1 mRNA. Our finding provides new insights into the mechanism of tumor angiogenesis. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Metabolic characterization of imatinib-resistant BCR-ABL T315I chronic myeloid leukemia cells indicates down-regulation of glycolytic pathway and low ROS production.

    Science.gov (United States)

    Ko, Byung Woong; Han, Jeongsu; Heo, Jun Young; Jang, Yunseon; Kim, Soo Jeong; Kim, Jungim; Lee, Min Joung; Ryu, Min Jeong; Song, Ik Chan; Jo, Young Suk; Kweon, Gi Ryang

    2016-09-01

    Long-term imatinib treatment induces drug-resistant chronic myeloid leukemia (CML) cells harboring T315I gate keeper mutation of breakpoint cluster region (BCR)-ABL oncogenic kinase. However, although cell proliferation is coupled with cellular energy status in CML carcinogenesis, the metabolic characteristics of T315I-mutant CML cells have never been investigated. Here, we analyzed cell proliferation activities and metabolic phenotypes, including cell proliferation, oxygen consumption, lactate production, and redox state in the KBM5 (imatinib-sensitive) and KBM5-T315I (imatinib-resistant) CML cell lines. Interestingly, KBM5-T315I cells showed decreased cell proliferation, lactate production, fatty acid synthesis, ROS production, and down regulation of mRNA expression related to ROS scavengers, such as SOD2, catalase, GCLm, and GPx1. Taken together, our data demonstrate that the lower growth ability of KBM5-T315I CML cells might be related to the decreased expression of glycolysis-related genes and ROS levels, and this will be used to identify therapeutic targets for imatinib resistance in CML.

  5. Regulation of extracellular polygalacturonase production in Pseudomonas solanacearum. Progress report, [May 1, 1992--April 30, 1994

    Energy Technology Data Exchange (ETDEWEB)

    Allen, C.

    1994-06-01

    Pseudomonas solanacearum is an economically important plant pathogen that causes bacterial wilt disease of diverse crops. The bacterium produces at least three isozymes of polygalacturonase, which degrade plant cell walls and contribute substantially to bacterial wilt disease development. The central objective of this research project is to determine how expression of these enzymes is regulated. To this end, we isolated a positive trans-acting regulator of polygalacturonase production (pehR). We have focused on further characterization of the pehR mutant pheonotype, and studies of pehR expression. Preliminary results suggest pehR also regulates bacterial motility. An investigation of two unusual tyrosine phosphoproteins in P. solanacearum is also described.

  6. World integrated medicine forum on the regulation of homoeopathic medicinal products: National and global strategies

    Directory of Open Access Journals (Sweden)

    Harleen Kaur

    2017-01-01

    Full Text Available The first World Integrated Medicine Forum on the regulation of homoeopathic medicinal products included 50 delegates from 25 countries to discuss the current state of regulation of homoeopathic medicinal products (HMPs. The stakeholders in attendance included government officials, manufacturers, pharmacopoeia organisations, pharmacists, and healthcare providers worldwide. The Minister of Ayurveda, Yoga, Unani, Siddha and Homoeopathy from India, who is likely the only high ranking national official in the world specifically dedicated to oversight of traditional medicines including Homoeopathy, was the keynote speaker for the meeting. The core presentations delivered information on the country-to-country variance in regulatory requirements for homoeopathic medicine manufacture and marketing. Different speakers addressed the current pharmacopeia structures in various countries, variance in premarket approval process, regulatory frameworks for homoeopathic medicines, labelling requirements, safety requirements, marketing approaches and good manufacturing practices. Debates focused on quality control testing, stability of intermediate stocks, shelf life of finished products, pre-market approval process and labelling and they shed light on regional differences in regulation. A lengthy discussion was held on the potential value of harmonisation of pharmacopoeias, manufacturing standards, safety evaluation and labelling. The group consensus was to meet again to pursue specific topics. Daily summaries of take-away points are provided at the end of each day's talk summaries. Much acclaim was won by the organisers for materialising this unique forum which proved to be an apt platform for rigorous discussions on lesser discussed, but very vital points such as regulations of HMPs, harmonisation of pharmacopeias and linking industry and regulators' sectors for unified efforts for global development of Homoeopathy.

  7. Cyclin E-p27 opposition and regulation of the G1 phase of the cell cycle in the murine neocortical PVE: a quantitative analysis of mRNA in situ hybridization

    Science.gov (United States)

    Delalle, I.; Takahashi, T.; Nowakowski, R. S.; Tsai, L. H.; Caviness, V. S. Jr

    1999-01-01

    We have analyzed the expression patterns of mRNAs of five cell cycle related proteins in the ventricular zone of the neocortical cerebral wall over the course of the neuronogenetic interval in the mouse. One set of mRNAs (cyclin E and p21) are initially expressed at high levels but expression then falls to a low asymptote. A second set (p27, cyclin B and cdk2) are initially expressed at low levels but ascend to peak levels only to decline again. These patterns divide the overall neuronogenetic interval into three phases. In phase 1 cyclin E and p21 levels of mRNA expression are high, while those of mRNAs of p27, cdk2 and cyclin B are low. In this phase the fraction of cells leaving the cycle after each mitosis, Q, is low and the duration of the G1 phase, TG1, is short. In phase 2 levels of expression of cyclin E and p21 fall to asymptote while levels of expression of mRNA of the other three proteins reach their peaks. Q increases to approach 0.5 and TG1 increases even more rapidly to approach its maximum length. In phase 3 levels of expression of cyclin E and p21 mRNAs remain low and those of the mRNAs of the other three proteins fall. TG1 becomes maximum and Q rapidly increases to 1.0. The character of these phases can be understood in part as consequences of the reciprocal regulatory influence of p27 and cyclin E and of the rate limiting functions of p27 at the restriction point and of cyclin E at the G1 to S transition.

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

    Science.gov (United States)

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

    2015-01-01

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

  9. Modulation of estrogen receptor mRNA expression by melatonin in MCF-7 human breast cancer cells.

    Science.gov (United States)

    Molis, T M; Spriggs, L L; Hill, S M

    1994-12-01

    Melatonin, the hormonal product of the pineal gland, has been shown to inhibit the development of mammary tumors in vivo and the proliferation of MCF-7 human breast cancer cells in vitro by mechanisms not yet identified. However, previous studies have demonstrated that melatonin significantly decreased estrogen-binding activity and the expression of immunoreactive estrogen receptor (ER) in MCF-7 breast cancer cells. To determine the mechanism(s) by which melatonin regulates ER expression in MCF-7 cells, the relationship between the level of steady state ER mRNA and the rate of ER gene transcription were examined in response to melatonin. Physiological concentrations of melatonin decreased steady state levels of ER mRNA expression in a dose- and time-specific manner. This decrease was not dependent upon the presence of estrogen since similar decreases in steady state ER mRNA levels were seen in MCF-7 cells cultured in both complete and estrogen-depleted media. The decreased expression of ER mRNA in response to melatonin appears to be directly related to the suppression of transcription of the ER gene. This regulation is independent of the synthesis of new proteins, as cycloheximide was unable to block the melatonin-induced decrease of steady-state ER mRNA levels. The down-regulation of ER by melatonin appears to not be mediated via a direct interaction with the ER and subsequent feedback on its own expression, since melatonin treatment did not alter the transcriptional regulatory ability of the fully activated wild type ER or a constitutively active hormone-binding domain-deleted ER variant. In addition, the stability of the ER transcript was unaffected by melatonin. Thus, it appears that the antiproliferative actions of this pineal indoleamine are mediated, at least in part, through the suppression of the transcription of the ER gene in MCF-7 human breast cancer cells.

  10. A SWI/SNF Chromatin Remodelling Protein Controls Cytokinin Production through the Regulation of Chromatin Architecture

    KAUST Repository

    Jégu, Teddy

    2015-10-12

    Chromatin architecture determines transcriptional accessibility to DNA and consequently gene expression levels in response to developmental and environmental stimuli. Recently, chromatin remodelers such as SWI/SNF complexes have been recognized as key regulators of chromatin architecture. To gain insight into the function of these complexes during root development, we have analyzed Arabidopsis knock-down lines for one sub-unit of SWI/SNF complexes: BAF60. Here, we show that BAF60 is a positive regulator of root development and cell cycle progression in the root meristem via its ability to down-regulate cytokinin production. By opposing both the deposition of active histone marks and the formation of a chromatin regulatory loop, BAF60 negatively regulates two crucial target genes for cytokinin biosynthesis (IPT3 and IPT7) and one cell cycle inhibitor (KRP7). Our results demonstrate that SWI/SNF complexes containing BAF60 are key factors governing the equilibrium between formation and dissociation of a chromatin loop controlling phytohormone production and cell cycle progression.

  11. Mapping medical marijuana: state laws regulating patients, product safety, supply chains and dispensaries, 2017.

    Science.gov (United States)

    Klieger, Sarah B; Gutman, Abraham; Allen, Leslie; Pacula, Rosalie Liccardo; Ibrahim, Jennifer K; Burris, Scott

    2017-12-01

    (1) To describe open source legal data sets, created for research use, that capture the key provisions of US state medical marijuana laws. The data document how state lawmakers have regulated a medicine that remains, under federal law, a Schedule I illegal drug with no legitimate medical use. (2) To demonstrate the variability that exists across states in rules governing patient access, product safety and dispensary practice. Two legal researchers collected and coded state laws governing marijuana patients, product safety and dispensaries in effect on 1 February 2017, creating three empirical legal data sets. We used summary tables to identify the variation in specific statutory provisions specified in each state's medical marijuana law as it existed on 1 February 2017. We compared aspects of these laws to the traditional Federal approach to regulating medicine. Full data sets, codebooks and protocols are available through the Prescription Drug Abuse Policy System (http://www.pdaps.org/; Archived at http://www.webcitation.org/6qv5CZNaZ on 2 June 2017). Twenty-eight states (including the District of Columbia) have authorized medical marijuana. Twenty-seven specify qualifying diseases, which differ across states. All states protect patient privacy; only 14 protect patients against discrimination. Eighteen states have mandatory product safety testing before any sale. While the majority have package/label regulations, states have a wide range of specific requirements. Most regulate dispensaries (25 states), with considerable variation in specific provisions such as permitted product supply sources number of dispensaries per state and restricting proximity to various types of location. The federal ban in the United States on marijuana has resulted in a patchwork of regulatory strategies that are not uniformly consistent with the approach usually taken by the Federal government and whose effectiveness remains unknown. © 2017 Society for the Study of Addiction.

  12. Comparative Analysis of mRNA Isoform Expression in Cardiac Hypertrophy and Development Reveals Multiple Post-Transcriptional Regulatory Modules

    Science.gov (United States)

    Park, Ji Yeon; Li, Wencheng; Zheng, Dinghai; Zhai, Peiyong; Zhao, Yun; Matsuda, Takahisa; Vatner, Stephen F.; Sadoshima, Junichi; Tian, Bin

    2011-01-01

    Cardiac hypertrophy is enlargement of the heart in response to physiological or pathological stimuli, chiefly involving growth of myocytes in size rather than in number. Previous studies have shown that the expression pattern of a group of genes in hypertrophied heart induced by pressure overload resembles that at the embryonic stage of heart development, a phenomenon known as activation of the “fetal gene program”. Here, using a genome-wide approach we systematically defined genes and pathways regulated in short- and long-term cardiac hypertrophy conditions using mice with transverse aortic constriction (TAC), and compared them with those regulated at different stages of embryonic and postnatal development. In addition, exon-level analysis revealed widespread mRNA isoform changes during cardiac hypertrophy resulting from alternative usage of terminal or internal exons, some of which are also developmentally regulated and may be attributable to decreased expression of Fox-1 protein in cardiac hypertrophy. Genes with functions in certain pathways, such as cell adhesion and cell morphology, are more likely to be regulated by alternative splicing. Moreover, we found 3′UTRs of mRNAs were generally shortened through alternative cleavage and polyadenylation in hypertrophy, and microRNA target genes were generally de-repressed, suggesting coordinated mechanisms to increase mRNA stability and protein production during hypertrophy. Taken together, our results comprehensively delineated gene and mRNA isoform regulation events in cardiac hypertrophy and revealed their relations to those in development, and suggested that modulation of mRNA isoform expression plays an importance role in heart remodeling under pressure overload. PMID:21799842

  13. Effect of Food Regulation on the Spanish Food Processing Industry: A Dynamic Productivity Analysis.

    Directory of Open Access Journals (Sweden)

    Magdalena Kapelko

    Full Text Available This article develops the decomposition of the dynamic Luenberger productivity growth indicator into dynamic technical change, dynamic technical inefficiency change and dynamic scale inefficiency change in the dynamic directional distance function context using Data Envelopment Analysis. These results are used to investigate for the Spanish food processing industry the extent to which dynamic productivity growth and its components are affected by the introduction of the General Food Law in 2002 (Regulation (EC No 178/2002. The empirical application uses panel data of Spanish meat, dairy, and oils and fats industries over the period 1996-2011. The results suggest that in the oils and fats industry the impact of food regulation on dynamic productivity growth is negative initially and then positive over the long run. In contrast, the opposite pattern is observed for the meat and dairy processing industries. The results further imply that firms in the meat processing and oils and fats industries face similar impacts of food safety regulation on dynamic technical change, dynamic inefficiency change and dynamic scale inefficiency change.

  14. Effect of Food Regulation on the Spanish Food Processing Industry: A Dynamic Productivity Analysis

    Science.gov (United States)

    Kapelko, Magdalena; Lansink, Alfons Oude; Stefanou, Spiro E.

    2015-01-01

    This article develops the decomposition of the dynamic Luenberger productivity growth indicator into dynamic technical change, dynamic technical inefficiency change and dynamic scale inefficiency change in the dynamic directional distance function context using Data Envelopment Analysis. These results are used to investigate for the Spanish food processing industry the extent to which dynamic productivity growth and its components are affected by the introduction of the General Food Law in 2002 (Regulation (EC) No 178/2002). The empirical application uses panel data of Spanish meat, dairy, and oils and fats industries over the period 1996-2011. The results suggest that in the oils and fats industry the impact of food regulation on dynamic productivity growth is negative initially and then positive over the long run. In contrast, the opposite pattern is observed for the meat and dairy processing industries. The results further imply that firms in the meat processing and oils and fats industries face similar impacts of food safety regulation on dynamic technical change, dynamic inefficiency change and dynamic scale inefficiency change. PMID:26057878

  15. Effect of Food Regulation on the Spanish Food Processing Industry: A Dynamic Productivity Analysis.

    Science.gov (United States)

    Kapelko, Magdalena; Oude Lansink, Alfons; Stefanou, Spiro E

    2015-01-01

    This article develops the decomposition of the dynamic Luenberger productivity growth indicator into dynamic technical change, dynamic technical inefficiency change and dynamic scale inefficiency change in the dynamic directional distance function context using Data Envelopment Analysis. These results are used to investigate for the Spanish food processing industry the extent to which dynamic productivity growth and its components are affected by the introduction of the General Food Law in 2002 (Regulation (EC) No 178/2002). The empirical application uses panel data of Spanish meat, dairy, and oils and fats industries over the period 1996-2011. The results suggest that in the oils and fats industry the impact of food regulation on dynamic productivity growth is negative initially and then positive over the long run. In contrast, the opposite pattern is observed for the meat and dairy processing industries. The results further imply that firms in the meat processing and oils and fats industries face similar impacts of food safety regulation on dynamic technical change, dynamic inefficiency change and dynamic scale inefficiency change.

  16. A novel role for IkappaBzeta in the regulation of IFNgamma production.

    Directory of Open Access Journals (Sweden)

    Raquel M Raices

    2009-08-01

    Full Text Available IkappaBzeta is a novel member of the IkappaB family of NFkappaB regulators, which modulates NFkappaB activity in the nucleus, rather than controlling its nuclear translocation. IkappaBzeta is specifically induced by IL-1beta and several TLR ligands and positively regulates NFkappaB-mediated transcription of genes such as IL-6 and NGAL as an NFkappaB binding co-factor. We recently reported that the IL-1 family cytokines, IL-1beta and IL-18, strongly synergize with TNFalpha for IFNgamma production in KG-1 cells, whereas the same cytokines alone have minimal effects on IFNgamma production. Given the striking similarities between the IL-1R and IL-18R signaling pathways we hypothesized that a common signaling event or gene product downstream of these receptors is responsible for the observed synergy. We investigated IkappaBzeta protein expression in KG-1 cells upon stimulation with IL-1beta, IL-18 and TNFalpha. Our results demonstrated that IL-18, as well as IL-1beta, induced moderate IkappaBzeta expression in KG-1 cells. However, TNFalpha synergized with IL-1beta and IL-18, whereas by itself it had a minimal effect on IkappaBzeta expression. NFkappaB inhibition resulted in decreased IL-1beta/IL-18/TNFalpha-stimulated IFNgamma release. Moreover, silencing of IkappaBzeta expression led to a specific decrease in IFNgamma production. Overall, our data suggests that IkappaBzeta positively regulates NFkappaB-mediated IFNgamma production in KG-1 cells.

  17. Two FERONIA-Like Receptor Kinases Regulate Apple Fruit Ripening by Modulating Ethylene Production

    Directory of Open Access Journals (Sweden)

    Meiru Jia

    2017-08-01

    Full Text Available Ethylene has long been known to be a critical signal controlling the ripening of climacteric fruits; however, the signaling mechanism underlying ethylene production during fruit development is unknown. Here, we report that two FERONIA-like receptor kinases (FERLs regulate fruit ripening by modulating ethylene production in the climacteric fruit, apple (Malus×domestica. Bioinformatic analysis indicated that the apple genome contains 14 members of the FER family (MdFERL1–17, of these 17 FERLs, MdFERL6 was expressed at the highest level in fruit. Heterologous expression of MdFERL6 or MdFERL1, the apple homolog of Arabidopsis FER, in another climacteric fruit, tomato (Solanum lycopersicum fruit delayed ripening and suppressed ethylene production. Overexpression and antisense expression of MdFERL6 in apple fruit calli inhibited and promoted ethylene production, respectively. Additionally, virus-induced gene silencing (VIGS of SlFERL1, the tomato homolog of FER, promoted tomato fruit ripening and ethylene production. Both MdFERL6 and MdFERL1 physically interacted with MdSAMS (S-adenosylmethionine synthase, a key enzyme in the ethylene biosynthesis pathway. MdFERL6 was expressed at high levels during early fruit development, but dramatically declined when fruit ripening commenced, implying that MdFERL6 might limit ethylene production prior to fruit development and the ethylene production burst during fruit ripening. These results indicate that FERLs regulate apple and tomato fruit ripening, shedding light on the molecular mechanisms underlying ripening in climacteric fruit.

  18. Two FERONIA-Like Receptor Kinases Regulate Apple Fruit Ripening by Modulating Ethylene Production.

    Science.gov (United States)

    Jia, Meiru; Du, Ping; Ding, Ning; Zhang, Qing; Xing, Sinian; Wei, Lingzhi; Zhao, Yaoyao; Mao, Wenwen; Li, Jizheng; Li, Bingbing; Jia, Wensuo

    2017-01-01

    Ethylene has long been known to be a critical signal controlling the ripening of climacteric fruits; however, the signaling mechanism underlying ethylene production during fruit development is unknown. Here, we report that two FERONIA-like receptor kinases (FERLs) regulate fruit ripening by modulating ethylene production in the climacteric fruit, apple (Malus×domestica). Bioinformatic analysis indicated that the apple genome contains 14 members of the FER family (MdFERL1-17), of these 17 FERLs, MdFERL6 was expressed at the highest level in fruit. Heterologous expression of MdFERL6 or MdFERL1, the apple homolog of Arabidopsis FER, in another climacteric fruit, tomato (Solanum lycopersicum) fruit delayed ripening and suppressed ethylene production. Overexpression and antisense expression of MdFERL6 in apple fruit calli inhibited and promoted ethylene production, respectively. Additionally, virus-induced gene silencing (VIGS) of SlFERL1, the tomato homolog of FER, promoted tomato fruit ripening and ethylene production. Both MdFERL6 and MdFERL1 physically interacted with MdSAMS (S-adenosylmethionine synthase), a key enzyme in the ethylene biosynthesis pathway. MdFERL6 was expressed at high levels during early fruit development, but dramatically declined when fruit ripening commenced, implying that MdFERL6 might limit ethylene production prior to fruit development and the ethylene production burst during fruit ripening. These results indicate that FERLs regulate apple and tomato fruit ripening, shedding light on the molecular mechanisms underlying ripening in climacteric fruit.

  19. Up-regulation of the receptor for advanced glycation end products in the skin biopsy specimens of patients with severe diabetic neuropathy.

    Science.gov (United States)

    Park, Su-Yeon; Kim, Young-A; Hong, Yoon-Ho; Moon, Min-Kyong; Koo, Bo-Kyeong; Kim, Tae Wan

    2014-10-01

    The receptor for advanced glycation end products (RAGE) may contribute to the development of diabetic neuropathy. To assess its relevance in humans, this study examined the expression of RAGE in the skin biopsy samples of patients with diabetes mellitus, and investigated its correlation with intraepidermal nerve-fiber density (IENFD) and clinical measures of neuropathy severity. Forty-four patients who either had type 2 diabetes or were prediabetes underwent clinical evaluation and a 3-mm skin punch biopsy. The clinical severity of their neuropathy was assessed using the Michigan Diabetic Neuropathy Score. IENFD was measured along with immunohistochemical staining for RAGE in 29 skin biopsy samples. The expression of RAGE was also quantified by real-time reverse-transcription PCR in the remaining 15 patients. RAGE was localized mostly in the dermal and subcutaneous vascular endothelia. The staining was more intense in patients with a lower IENFD (p=0.004). The quantity of RAGE mRNA was significantly higher in patients with severe neuropathy than in those with no or mild neuropathy (p=0.003). The up-regulation of RAGE was related to dyslipidemia and diabetic nephropathy. There was a trend toward decreased sural nerve action-potential amplitude and slowed peroneal motor-nerve conduction with increasing RAGE expression. The findings of this study demonstrate up-regulation of RAGE in skin biopsy samples from patients with diabetic neuropathy, supporting a pathogenic role of RAGE in the development of diabetic neuropathy.

  20. An R2R3-MYB Transcription Factor Regulates Eugenol Production in Ripe Strawberry Fruit Receptacles.

    Science.gov (United States)

    Medina-Puche, Laura; Molina-Hidalgo, Francisco Javier; Boersma, Maaike; Schuurink, Robert C; López-Vidriero, Irene; Solano, Roberto; Franco-Zorrilla, José-Manuel; Caballero, José Luis; Blanco-Portales, Rosario; Muñoz-Blanco, Juan

    2015-06-01

    Eugenol is a volatile phenylpropanoid that contributes to flower and ripe fruit scent. In ripe strawberry (Fragaria × ananassa) fruit receptacles, eugenol is biosynthesized by eugenol synthase (FaEGS2). However, the transcriptional regulation of this process is still unknown. We have identified and functionally characterized an R2R3 MYB transcription factor (emission of benzenoid II [FaEOBII]) that seems to be the orthologous gene of PhEOBII from Petunia hybrida, which contributes to the regulation of eugenol biosynthesis in petals. The expression of FaEOBII was ripening related and fruit receptacle specific, although high expression values were also found in petals. This expression pattern of FaEOBII correlated with eugenol content in both fruit receptacle and petals. The expression of FaEOBII was repressed by auxins and activated by abscisic acid, in parallel to the ripening process. In ripe strawberry receptacles, where the expression of FaEOBII was silenced, the expression of cinnamyl alcohol dehydrogenase1 and FaEGS2, two structural genes involved in eugenol production, was down-regulated. A subsequent decrease in eugenol content in ripe receptacles was also observed, confirming the involvement of FaEOBII in eugenol metabolism. Additionally, the expression of FaEOBII was under the control of FaMYB10, another R2R3 MYB transcription factor that regulates the early and late biosynthetic genes from the flavonoid/phenylpropanoid pathway. In parallel, the amount of eugenol in FaMYB10-silenced receptacles was also diminished. Taken together, these data indicate that FaEOBII plays a regulating role in the volatile phenylpropanoid pathway gene expression that gives rise to eugenol production in ripe strawberry receptacles. © 2015 American Society of Plant Biologists. All Rights Reserved.

  1. Chromatin landscaping in algae reveals novel regulation pathway for biofuels production

    Energy Technology Data Exchange (ETDEWEB)

    Ngan, Chew Yee; Wong, Chee-Hong; Choi, Cindy; Pratap, Abhishek; Han, James; Wei, Chia-Lin

    2013-02-19

    The diminishing reserve of fossil fuels calls for the development of biofuels. Biofuels are produced from renewable resources, including photosynthetic organisms, generating clean energy. Microalgae is one of the potential feedstock for biofuels production. It grows easily even in waste water, and poses no competition to agricultural crops for arable land. However, little is known about the algae lipid biosynthetic regulatory mechanisms. Most studies relied on the homology to other plant model organisms, in particular Arabidopsis or through low coverage expression analysis to identify key enzymes. This limits the discovery of new components in the biosynthetic pathways, particularly the genetic regulators and effort to maximize the production efficiency of algal biofuels. Here we report an unprecedented and de novo approach to dissect the algal lipid pathways through disclosing the temporal regulations of chromatin states during lipid biosynthesis. We have generated genome wide chromatin maps in chlamydomonas genome using ChIP-seq targeting 7 histone modifications and RNA polymerase II in a time-series manner throughout conditions activating lipid biosynthesis. To our surprise, the combinatory profiles of histone codes uncovered new regulatory mechanism in gene expression in algae. Coupled with matched RNA-seq data, chromatin changes revealed potential novel regulators and candidate genes involved in the activation of lipid accumulations. Genetic perturbation on these candidate regulators further demonstrated the potential to manipulate the regulatory cascade for lipid synthesis efficiency. Exploring epigenetic landscape in microalgae shown here provides powerful tools needed in improving biofuel production and new technology platform for renewable energy generation, global carbon management, and environmental survey.

  2. Product unconformable in the light of legal regulations and the ISO 9001:2000 standards

    Directory of Open Access Journals (Sweden)

    Justyna Górna

    2009-01-01

    Full Text Available The supervision of unconformable product is a key problem in the era of globalization. In Poland the supervision of safety product has been entrusted to the President of the Office of Competition and Consumer Protection. The agency supporting its activities is the Trade Inspection. Growing popularity of the quality systems conformable with the ISO 9000 standard caused that they have been included in the EU regulations as criteria of assessment of conformability with requirements. The certificate of the quality management system is indispensable for companies for functioning in many market areas. One should remember that quality management system will help the company to supervise unconformable products only when it really functions and is not just on paper. Only then it will function efficiently.

  3. Can green consumerism replace environmental regulation? A differentiated-products example

    Energy Technology Data Exchange (ETDEWEB)

    Eriksson, Clas [Department of Economics, Swedish University of Agricultural Sciences, P.O. Box 7013, S-750 07 Uppsala (Sweden)

    2004-09-01

    This paper assumes that consumers are willing to pay an extra premium for a good if it has a low impact on the environment. We examine if a little dose of such idealistic behavior has a large impact on the market equilibrium, and to what extent it can replace the environmental regulation. The analysis is carried out in a model with product differentiation, where consumers differ in their preferences for product quality. Consumers' willingness to pay the environmental premium may be uniformly or non-uniformly distributed. Green consumerism will only be modestly influential in both cases, despite the fact that product differentiation leads to relaxed competition and increased profits, and thereby creates leverage.

  4. Biofilm Formation by Bacillus subtilis Requires an Endoribonuclease-containing Multisubunit Complex that Controls mRNA Levels for the Matrix Gene Repressor SinR

    Science.gov (United States)

    DeLoughery, Aaron; Dengler, Vanina; Chai, Yunrong; Losick, Richard

    2016-01-01

    Biofilm formation by Bacillus subtilis is largely governed by a circuit in which the response regulator Spo0A turns on the gene for the anti-repressor SinI. SinI, in turn, binds to and inactivates SinR, a dedicated repressor of genes for matrix production. Mutants of the genes ylbF, ymcA, and yaaT are blocked in biofilm formation, but the mechanism by which they act has been mysterious. A recent report attributed their role in biofilm formation to stimulating Spo0A activity. However, we detect no measurable effect on the transcription of sinI. Instead, we find that the block in biofilm formation is caused by an increase in the levels of SinR and of its mRNA. Evidence is presented that YlbF, YmcA and YaaT interact with, and control the activity of, RNase Y, which is known to destabilize sinR mRNA. We show that the processing of another target of RNase Y, cggR-gapA mRNA, similarly depends on YlbF and YmcA. Our work suggests that sinR mRNA stability is an additional posttranscriptional control mechanism governing the switch to multicellularity and raises the possibility that YlbF, YmcA, and YaaT broadly regulate mRNA stability as part of an RNase Y-containing, multi-subunit complex. PMID:26434553

  5. Electron acceptor-based regulation of microbial greenhouse gas production from thawing permafrost

    Science.gov (United States)

    Bak, Ebbe; Jones, Eleanor; Yde, Jacob; Hodson, Andy; Mallon, Gunnar; Fisnter, Kai

    2017-04-01

    Permafrost contains about 35% of the global soil organic carbon (0-3 m depth). As a consequence of global warming, the active layer thickness is steadily increasing and its organic carbon is becoming available for degradation, causing a concomitant release of CO2 and CH4. The climate forcing feedbacks of permafrost thaw are determined by the rate of organic carbon degradation and to which degree it is released as CO2 or CH4. Methane is produced under anoxic conditions, but the factors that regulate its production are poorly constrained. In this study, we investigate how CH4 production is influenced by the presence of competing anaerobic processes with focus on the role of iron and sulfate reduction. We have collected permafrost cores to 2.2 meters depth from three different lowland sites in Adventdalen on Svalbard. From these cores, we have prepared anoxic batch incubation for each 25 cm depth interval and followed the production of CO2 and CH4 as well as the iron and sulfate reduction. This approach allows us to monitor the rate of the CO2 and CH4 production as well as to investigate the correlation between CH4 production and competing anaerobic respiration processes in the active layer as well in the permafrost. These investigations are accompanied by characterization of the carbon, iron and sulfate content in the soil and will be followed by characterization of the microbial community structure. The aim of this study is to get a better understanding of how the availability of sulfate and iron and the microbial community structure regulate the production of CO2 and CH4 in thawing permafrost, and to elucidate how the rate of the organic carbon degradation changes with depth in permafrost-affected soils. This study improves our understanding of climate feedback mechanisms operating during permafrost thaw.

  6. One carbon metabolism in anaerobic bacteria: Regulation of carbon and electron flow during organic acid production

    Energy Technology Data Exchange (ETDEWEB)

    Zeikus, J.G.; Jain, M.

    1993-12-31

    The project deals with understanding the fundamental biochemical mechanisms that physiologically control and regulate carbon and electron flow in anaerobic chemosynthetic bacteria that couple metabolism of single carbon compounds and hydrogen to the production of organic acids (formic, acetic, butyric, and succinic) or methane. The authors compare the regulation of carbon dioxide and hydrogen metabolism by fermentation, enzyme, and electron carrier analysis using Butyribacterium methylotrophicum, Anaeroblospirillum succiniciproducens, Methanosarcina barkeri, and a newly isolated tri-culture composed of a syntrophic butyrate degrader strain IB, Methanosarcina mazei and Methanobacterium formicicum as model systems. To understand the regulation of hydrogen metabolism during butyrate production or acetate degradation, hydrogenase activity in B. methylotrophicum or M. barkeri is measured in relation to growth substrate and pH; hydrogenase is purified and characterized to investigate number of hydrogenases; their localization and functions; and, their sequences are determined. To understand the mechanism for catabolic CO{sub 2} fixation to succinate the PEP carboxykinase enzyme and gene of A. succiniciproducens are purified and characterized. Genetically engineered strains of Escherichia coli containing the phosphoenolpyruvate (PEP) carboxykinase gene are examined for their ability to produce succinate in high yield. To understand the mechanism of fatty acid degradation by syntrophic acetogens during mixed culture methanogenesis formate and hydrogen production are characterized by radio tracer studies. It is intended that these studies provide strategies to improve anaerobic fermentations used for the production of organic acids or methane and, new basic understanding on catabolic CO{sub 2} fixation mechanisms and on the function of hydrogenase in anaerobic bacteria.

  7. Elevated mRNA expression of PGF2α receptor splice variant 2(FP-V2) in human decidua is associated with incomplete mifepristone-misoprostol-induced early medical abortion by regulation of interleukin-8.

    Science.gov (United States)

    Ma, Chao; Feng, Wenhua; Han, Weitian; Lu, Yongping; Liu, Wei; Sui, Yu; Zhao, Ning; Lye, Stephen J; Li, Jianxin

    2016-11-01

    The combination of mifepristone and misoprostol is an established method for the induction of early abortion, but 15% of women still experience the unpleasant side effect of incomplete medical abortion. The purpose of this study was to determine whether prostaglandin (PG) F2α receptor (FP) and its two isoforms (FP-V1 and FP-V2) in human decidua are associated incomplete abortion. Forty women who underwent medical abortion were recruited. Among them, there were 20 cases of incomplete abortion. The other 20 cases of complete abortion were used as controls. The expression levels of FP, FPV1 and FP-V2 in the decidua between of the two groups was detected by quantitative real-time polymerase chain reaction (PCR). Additionally, FP-V2 was knocked down using specific small interfering RNAs (siRNAs) in the primary cultures of decidual cells. The expression levels of cytokines in FP-V2 knockdown primary decidual cells and control decidual cells were detected by quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA). The FP and FP-V2 mRNA expression in the incomplete group was significantly higher than that in the complete group (p abortion and that IL-8 could be lined to this process.

  8. Optimal Regulation of the Balance between Productivity and Overwintering of Perennial Grasses in a Warmer Climate

    Directory of Open Access Journals (Sweden)

    Åshild Ergon

    2017-02-01

    Full Text Available Seasonal growth patterns of perennial plants are linked to patterns of acclimation and de-acclimation to seasonal stresses. The timing of cold acclimation (development of freezing resistance and leaf growth cessation in autumn, and the timing of de-acclimation and leaf regrowth in spring, is regulated by seasonal cues in the environment, mainly temperature and light factors. Warming will lead to new combinations of these cues in autumn and spring. Extended thermal growing seasons offer a possibility for obtaining increased yields of perennial grasses at high latitudes. Increased productivity in the autumn may not be possible in all high latitude regions due to the need for light during cold acclimation and the need for accumulating a carbohydrate storage prior to winter. There is more potential for increased yields in spring due to the availability of light, but higher probability of freezing events in earlier springs would necessitate a delay of de-acclimation, or an ability to rapidly re-acclimate. In order to optimize the balance between productivity and overwintering in the future, the regulation of growth and acclimation processes may have to be modified. Here, the current knowledge on the coordinated regulation of growth and freezing resistance in perennial grasses is reviewed.

  9. 4-Methylthio-3-butenyl isothiocyanate mediates nuclear factor (erythroid-derived 2)-like 2 activation by regulating reactive oxygen species production in human esophageal epithelial cells.

    Science.gov (United States)

    Hirata, Tadashi; Cho, Young-Man; Suzuki, Isamu; Toyoda, Takeshi; Akagi, Jun-Ichi; Nakamura, Yasushi; Numazawa, Satoshi; Ogawa, Kumiko

    2018-01-01

    4-Methylthio-3-butenyl isothiocyanate (MTBITC) extracted from daikon (Raphanus sativus), which shows antimutagenicity, may have applications as an effective chemopreventive agent in several cancers; however, few reports have described the associated mechanisms. We investigated whether MTBITC induced cytoprotective genes, including phase II enzymes, in Het-1A human esophageal epithelial cells. HMOX1, NQO1, and GCLC mRNA levels and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protein levels were increased in Het-1A cells treated with 10 μM MTBITC. Reactive oxygen species (ROS) tended to increase when Het-1A cells were treated with MTBITC, and the increases in ROS and Nrf2 expression in the cells treated with MTBITC were completely abolished by treatment with N-acetyl-l-cysteine. We also examined the relationships between Nrf2 activation and mitogen-activated protein kinase (MAPK) signaling by western blot analysis. MTBITC induced extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 phosphorylation in Het-1A cells; however, MTBITC did not affect the relationship between Nrf2 activation and MAPK responses. In the present study, we found that MTBITC induced Nrf2 activation and cytoprotective genes via ROS production in Het-1A cells. These results suggest that MTBITC may have the potential for preventing esophageal carcinogenesis through modification of carcinogen metabolism by phase II enzyme induction via ROS production. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. [A contribution to the development of advertising in pharmacy II. Historical development of regulation of advertising of medicinal products].

    Science.gov (United States)

    Vranová, Vilma

    2012-10-01

    The article deals with the development of regulation of advertising of medicinal products in the Czech Lands of the Habsburg Monarchy and Czechoslovakia in the years 1775-1938. Advertising medicines had and has its specifics and its regulation had been addressed by specific standards and linked to other health laws and regulations. Regulation of advertising of medicinal products has undergone a long process from the initial total ban on advertising to the establishment of clear rules, some of which, such as restrictions on advertising prescription-only medicines only to the professional healthcare press, are still valid.

  11. Insights into the global regulation of anaerobic metabolism for improved biohydrogen production.

    Science.gov (United States)

    Lu, Yuan; Zhao, Hongxin; Zhang, Chong; Xing, Xin-Hui

    2016-01-01

    To improve the biohydrogen yield in bacterial dark fermentation, a new approach of global anaerobic regulation was introduced. Two cellular global regulators FNR and NarP were overexpressed in two model organisms: facultatively anaerobic Enterobacter aerogenes (Ea) and strictly anaerobic Clostridium paraputrificum (Cp). The overexpression of FNR and NarP greatly altered anaerobic metabolism and increased the hydrogen yield by 40%. Metabolic analysis showed that the global regulation caused more reducing environment inside the cell. To get a thorough understanding of the global metabolic regulation, more genes (fdhF, fhlA, ppk, Cb-fdh1, and Sc-fdh1) were overexpressed in different Ea and Cp mutants. For the first time, it demonstrated that there were approximately linear relationships between the relative change of hydrogen yield and the relative change of NADH yield or ATP yield. It implied that cellular reducing power and energy level played vital roles in the biohydrogen production. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. The safety and regulation of natural products used as foods and food ingredients.

    Science.gov (United States)

    Abdel-Rahman, Ali; Anyangwe, Njwen; Carlacci, Louis; Casper, Steve; Danam, Rebecca P; Enongene, Evaristus; Erives, Gladys; Fabricant, Daniel; Gudi, Ramadevi; Hilmas, Corey J; Hines, Fred; Howard, Paul; Levy, Dan; Lin, Ying; Moore, Robert J; Pfeiler, Erika; Thurmond, T Scott; Turujman, Saleh; Walker, Nigel J

    2011-10-01

    The use of botanicals and dietary supplements derived from natural substances as an adjunct to an improved quality of life or for their purported medical benefits has become increasingly common in the United States. This review addresses the safety assessment and regulation of food products containing these substances by the U.S. Food and Drug Administration (FDA). The issue of safety is particularly critical given how little information is available on the toxicity of some of these products. The first section uses case studies for stevia and green tea extracts as examples of how FDA evaluates the safety of botanical and herbal products submitted for consideration as Generally Recognized as Safe under the Federal Food, Drug, and Cosmetics Act. The 1994 Dietary Supplement Health Education Act (DSHEA) created a regulatory framework for dietary supplements. The article also discusses the regulation of this class of dietary supplements under DSHEA and addresses the FDA experience in analyzing the safety of natural ingredients described in pre-market safety submissions. Lastly, we discuss an ongoing interagency collaboration to conduct safety testing of nominated dietary supplements.

  13. [Food supplements on the Hungarian market: regulations of marketing and of the composition of the products].

    Science.gov (United States)

    Lugasi, Andrea; Horacsek, Márta; Martos, Eva

    2010-09-26

    According to recent legislation, food supplements are foodstuffs with the purpose of supplementing normal diet. Food supplements are concentrated sources of nutrients such as vitamins and minerals and other substances with a physiological or nutritional effect. In Hungary, marketing of food supplements has not been bound to pre-market authorization since joining to the European Union. The food business operator, who is responsible for production or distribution of the product, must notify it at National Institute for Food and Nutrition Science latest at the time when the product has been placed on the market and it can be distributed simultaneously. Distribution, ingredients, and all those information which appear on the label are determined by numerous regulations and prescriptions but at the same time the lack of harmonized legislation at certain places may cause a lot of problems on Community level. The first part of the study shows the laws and regulations influencing the distribution and ingredients of food supplements, while the main target of the second part is to introduce the evaluation process of components from nutritional and physiological point of view, and the role played by the food supplements in nutrition.

  14. Basic Science and Public Policy: Informed Regulation for Nicotine and Tobacco Products.

    Science.gov (United States)

    Fowler, Christie D; Gipson, Cassandra D; Kleykamp, Bethea A; Rupprecht, Laura E; Harrell, Paul T; Rees, Vaughan W; Gould, Thomas J; Oliver, Jason; Bagdas, Deniz; Damaj, M Imad; Schmidt, Heath D; Duncan, Alexander; De Biasi, Mariella

    2017-08-03

    Scientific discoveries over the past few decades have provided significant insight into the abuse liability and negative health consequences associated with tobacco and nicotine-containing products. While many of these advances have led to the development of policies and laws that regulate access to and formulations of these products, further research is critical to guide future regulatory efforts, especially as novel nicotine-containing products are introduced and selectively marketed to vulnerable populations. In this narrative review, we provide an overview of the scientific findings that have impacted regulatory policy and discuss considerations for further translation of science into policy decisions. We propose that open, bidirectional communication between scientists and policy makers is essential to develop transformative preventive- and intervention-focused policies and programs to reduce appeal, abuse liability, and toxicity of the products. Through these types of interactions, collaborative efforts to inform and modify policy have the potential to significantly decrease the use of tobacco and alternative nicotine products and thus enhance health outcomes for individuals. This work addresses current topics in the nicotine and tobacco research field to emphasize the importance of basic science research and provide examples of how it can be utilized to inform public policy. In addition to relaying current thoughts on the topic from experts in the field, the article encourages continued efforts and communication between basic scientists and policy officials.

  15. REGULATION OF PRODUCT CERTIFICATION AND STANDARDIZATION PROCESSES IN THE EUROPEAN UNION

    Directory of Open Access Journals (Sweden)

    Liudmyla Shabelnyk

    2016-11-01

    Full Text Available Research is aimed at investigation of current state of technical regulation system in European Union. Special attention is paid to peculiarities of standardization and certification in certain countries of EU in order to implement foreign experience in Ukraine. Despite the wide academic coverage of technical regulation issue, a range of issues remain unsolved, especially the problem of historical retrospective and possibility of positive experience implementation. Subject of study. Theoretical and methodological approaches to the regulation of certification and standardization of processes in the EU, as well as comparative analysis of the systems in the past and the present. Methodology. The survey is based on research of available publications on the topic, comparative analysis, induction and deduction. Issues of standardization and certification were widely discovered by numerous researchers: F.D. Duyvis (1960, W. Hesser and A. Inklaar (1998, C. Shapiro (1999, A. Richen and A. Steinhorst (2005. But in spite of the deep theoretical background of scientific works, it is obvious that the most important documents for technical regulations are ISO standards (especially ISO/ IEC 17021:2006 «Conformity assessment – Requirements for bodies providing audit and certification of management systems». Results. The article discovers relationship between main standardization bodies in EU, studies main legislative documents, reveals its historical retrospective and provides in-depth study of technical regulation system in Germany and the UK. Information from the article can be used for general understanding of main processes of technical regulation in EU; practical discovery of marking signs on the products and comparative analysis of standardization systems in different countries. Practical implications. It is important to mention practical demonstration of technical requirements in the EU: the signs marking Certificate IN, Certificate VS, Certificate

  16. Protein targeting to subcellular organelles via MRNA localization.

    Science.gov (United States)

    Weis, Benjamin L; Schleiff, Enrico; Zerges, William

    2013-02-01

    Cells have complex membranous organelles for the compartmentalization and the regulation of most intracellular processes. Organelle biogenesis and maintenance requires newly synthesized proteins, each of which needs to go from the ribosome translating its mRNA to the correct membrane for insertion or transclocation to an a organellar subcompartment. Decades of research have revealed how proteins are targeted to the correct organelle and translocated across one or more organelle membranes ro the compartment where they function. The paradigm examples involve interactions between a peptide sequence in the protein, localization factors, and various membrane embedded translocation machineries. Membrane translocation is either cotranslational or posttranslational depending on the protein and target organelle. Meanwhile research in embryos, neurons and yeast revealed an alternative targeting mechanism in which the mRNA is localized and only then translated to synthesize the protein in the correct location. In these cases, the targeting information is coded by the cis-acting sequences in the mRNA ("Zipcodes") that interact with localization factors and, in many cases, are transported by the molecular motors on the cytoskeletal filaments. Recently, evidence has been found for this "mRNA based" mechanism in organelle protein targeting to endoplasmic reticulum, mitochondria, and the photosynthetic membranes within chloroplasts. Here we review known and potential roles of mRNA localization in protein targeting to and within organelles. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.

  17. TGF-β1 regulation of estrogen production in mature rat Leydig cells.

    Directory of Open Access Journals (Sweden)

    Man-Li Liu

    Full Text Available BACKGROUND: Besides androgens, estrogens produced in Leydig cells are also crucial for mammalian germ cell differentiation. Transforming growth factor-β1 (TGF-β1 is now known to have multiple effects on regulation of Leydig cell function. The objective of the present study is to determine whether TGF-β1 regulates estradiol (E2 synthesis in adult rat Leydig cells and then to assess the impact of TGF-β1 on Cx43-based gap junctional intercellular communication (GJIC between Leydig cells. METHODOLOGY/PRINCIPAL FINDINGS: Primary cultured Leydig cells were incubated in the presence of recombinant TGF-β1 and the production of E2 as well as testosterone (T were measured by RIA. The activity of P450arom was addressed by the tritiated water release assay and the expression of Cyp19 gene was evaluated by Western blotting and real time RT-PCR. The expression of Cx43 and GJIC were investigated with immunofluorescence and fluorescence recovery after photo-bleaching (FRAP, respectively. Results from this study show that TGF-β1 down-regulates the level of E2 secretion and the activity of P450arom in a dose-dependent manner in adult Leydig cells. In addition, the expression of Cx43 and GJIC was closely related to the regulation of E2 and TGF-β1, and E2 treatment in turn restored the inhibition of TGF-β1 on GJIC. CONCLUSIONS: Our results indicate, for the first time in adult rat Leydig cells, that TGF-β1 suppresses P450arom activity, as well as the expression of the Cyp19 gene, and that depression of E2 secretion leads to down-regulation of Cx43-based GJIC between Leydig cells.

  18. Circadian Clock genes Per2 and clock regulate steroid production, cell proliferation, and luteinizing hormone receptor transcription in ovarian granulosa cells

    Energy Technology Data Exchange (ETDEWEB)

    Shimizu, Takashi, E-mail: shimizut@obihiro.ac.jp [Graduate School of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555 (Japan); Hirai, Yuko; Murayama, Chiaki; Miyamoto, Akio [Graduate School of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555 (Japan); Miyazaki, Hitoshi [Gene Research Center, University of Tsukuba, Tsukuba, Ibaraki 305-8572 (Japan); Miyazaki, Koyomi [Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST) Central 6, 1-1-1, Higashi, Tsukuba, Ibaraki 305-8566 (Japan)

    2011-08-19

    Highlights: {yields} Treatment with Per2 and Clock siRNAs decreased the number of granulosa cells and LHr expression. {yields}Per2 siRNA treatment did not stimulate the production of estradiol and expression of P450arom. {yields} Clock siRNA treatment inhibited the production of estradiol and expression of P450arom mRNA. {yields}Per2 and Clock siRNA treatment increased and unchanged, respectively, progesterone production in FSH-treated granulosa cells. {yields} The expression of StAR mRNA was increased by Per2 siRNA and unchanged by Clock siRNA. -- Abstract: Circadian Clock genes are associated with the estrous cycle in female animals. Treatment with Per2 and Clock siRNAs decreased the number of granulosa cells and LHr expression in follicle-stimulating hormone FSH-treated granulosa cells. Per2 siRNA treatment did not stimulate the production of estradiol and expression of P450arom, whereas Clock siRNA treatment inhibited the production of estradiol and expression of P450arom mRNA. Per2 and Clock siRNA treatment increased and unchanged, respectively, progesterone production in FSH-treated granulosa cells. Similarly, expression of StAR mRNA was increased by Per2 siRNA and unchanged by Clock siRNA. Our data provide a new insight that Per2 and Clock have different action on ovarian granulosa cell functions.

  19. Potential utility of natural products as regulators of breast cancer-associated aromatase promoters

    Directory of Open Access Journals (Sweden)

    Walker Larry A

    2011-06-01

    Full Text Available Abstract Aromatase, the key enzyme in estrogen biosynthesis, converts androstenedione to estrone and testosterone to estradiol. The enzyme is expressed in various tissues such as ovary, placenta, bone, brain, skin, and adipose tissue. Aromatase enzyme is encoded by a single gene CYP 19A1 and its expression is controlled by tissue-specific promoters. Aromatase mRNA is primarily transcribed from promoter I.4 in normal breast tissue and physiological levels of aromatase are found in breast adipose stromal fibroblasts. Under the conditions of breast cancer, as a result of the activation of a distinct set of aromatase promoters (I.3, II, and I.7 aromatase expression is enhanced leading to local overproduction of estrogen that promotes breast cancer. Aromatase is considered as a potential target for endocrine treatment of breast cancer but due to nonspecific reduction of aromatase activity in other tissues, aromatase inhibitors (AIs are associated with undesirable side effects such as bone loss, and abnormal lipid metabolism. Inhibition of aromatase expression by inactivating breast tumor-specific aromatase promoters can selectively block estrogen production at the tumor site. Although several synthetic chemical compounds and nuclear receptor ligands are known to inhibit the activity of the tumor-specific aromatase promoters, further development of more specific and efficacious drugs without adverse effects is still warranted. Plants are rich in chemopreventive agents that have a great potential to be used in chemotherapy for hormone dependent breast cancer which could serve as a source for natural AIs. In this brief review, we summarize the studies on phytochemicals such as biochanin A, genistein, quercetin, isoliquiritigenin, resveratrol, and grape seed extracts related to their effect on the activation of breast cancer-associated aromatase promoters and discuss their aromatase inhibitory potential to be used as safer chemotherapeutic agents for

  20. Coupling mRNA processing with transcription in time and space.

    Science.gov (United States)

    Bentley, David L

    2014-03-01

    Maturation of mRNA precursors often occurs simultaneously with their synthesis by RNA polymerase II (Pol II). The co-transcriptional nature of mRNA processing has permitted the evolution of coupling mechanisms that coordinate transcription with mRNA capping, splicing, editing and 3' end formation. Recent experiments using sophisticated new methods for analysis of nascent RNA have provided important insights into the relative amount of co-transcriptional and post-transcriptional processing, the relationship between mRNA elongation and processing, and the role of the Pol II carboxy-terminal domain (CTD) in regulating these processes.

  1. 78 FR 29263 - Rules andRegulations Under the Textile Fiber Products Identification Act

    Science.gov (United States)

    2013-05-20

    ...Based on comments received in response to its Advance Notice of Proposed Rulemaking (``ANPR''), the Commission proposes amending the rules and regulations under the Textile Fiber Products Identification Act (``Textile Rules'' or ``Rules'') to: Incorporate the updated ISO standard 2076:2010(E); allow certain hang-tags that do not disclose the product's full fiber content information; better address electronic commerce by amending the definition of the terms invoice and invoice or other paper; update the guaranty provisions by, among other things, replacing the requirement that suppliers provide a guaranty signed under penalty of perjury with a certification that must be renewed annually, and revising accordingly the form used to file continuing guaranties with the Commission under the Textile, Fur, and Wool Acts; and clarify several other provisions. The Commission seeks comment on these proposals and several remaining issues.

  2. A Human Variant of Glucose-Regulated Protein 94 That Inefficiently Supports IGF Production

    DEFF Research Database (Denmark)

    Marzec, Michal; Hawkes, Colin P; Eletto, Davide

    2016-01-01

    IGFs are critical for normal intrauterine and childhood growth and sustaining health throughout life. We showed previously that the production of IGF-1 and IGF-2 requires interaction with the chaperone glucose-regulated protein 94 (GRP94) and that the amount of secreted IGFs is proportional...... to the GRP94 activity. Therefore, we tested the hypothesis that functional polymorphisms of human GRP94 affect IGF production and thereby human health. We describe a hypomorphic variant of human GRP94, P300L, whose heterozygous carriers have 9% lower circulating IGF-1 concentration. P300L was found first...... in a child with primary IGF deficiency and was later shown to be a noncommon single-nucleotide polymorphism with frequencies of 1%-4% in various populations. When tested in the grp94(-/-) cell-based complementation assay, P300L supported only approximately 58% of IGF secretion relative to wild-type GRP94...

  3. Increasing Malonyl-CoA Derived Product through Controlling the Transcription Regulators of Phospholipid Synthesis in Saccharomyces cerevisiae.

    Science.gov (United States)

    Chen, Xiaoxu; Yang, Xiaoyu; Shen, Yu; Hou, Jin; Bao, Xiaoming

    2017-05-19

    Malonyl-CoA is a precursor of a variety of compounds such as polyketides and flavonoids. In Saccharomyces cerevisiae, malonyl-CoA concentration is tightly regulated and therefore maintained at a very low level, limiting the production of malonyl-CoA-derived chemicals. Here we manipulated the phospholipid synthesis transcriptional regulators to control the malonyl-CoA levels and increase the downstream product. Through manipulating different regulators including Ino2p, Ino4p, Opi1p, and a series of synthetic Ino2p variants, combining with studying the inositol and choline effect, the engineered strain achieved a 9-fold increase of the titer of malonyl-CoA-derived product 3-hydroxypropionic acid, which is among the highest improvement relative to previously reported strategies. Our study provides a new strategy to regulate malonyl-CoA availability and will contribute to the production of other highly valued malonyl-CoA-derived chemicals.

  4. Identification of BCAP-{sub L} as a negative regulator of the TLR signaling-induced production of IL-6 and IL-10 in macrophages by tyrosine phosphoproteomics

    Energy Technology Data Exchange (ETDEWEB)

    Matsumura, Takayuki [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, Shinjuku-ku, Tokyo 162-0041 (Japan); Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku-ku, Tokyo 162-8480 (Japan); Oyama, Masaaki; Kozuka-Hata, Hiroko [Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639 (Japan); Ishikawa, Kosuke; Inoue, Takafumi [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, Shinjuku-ku, Tokyo 162-0041 (Japan); Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku-ku, Tokyo 162-8480 (Japan); Muta, Tatsushi [Laboratory of Cell Recognition and Response, Graduate School of Life Sciences, Tohoku University, Sendai 980-8578 (Japan); Semba, Kentaro, E-mail: ksemba@waseda.jp [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, Shinjuku-ku, Tokyo 162-0041 (Japan); Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku-ku, Tokyo 162-8480 (Japan); Inoue, Jun-ichiro, E-mail: jun-i@ims.u-tokyo.ac.jp [Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639 (Japan); Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639 (Japan)

    2010-09-17

    Research highlights: {yields} Twenty five tyrosine-phosphorylated proteins in LPS-stimulated macrophages were determined. {yields} BCAP is a novel tyrosine-phosphorylated protein in LPS-stimulated macrophages. {yields} BCAP-{sub L} inhibits IL-6 and IL-10 production in LPS-stimulated macrophages. -- Abstract: Toll-like receptor (TLR) signaling in macrophages is essential for anti-pathogen responses such as cytokine production and antigen presentation. Although numerous reports suggest that protein tyrosine kinases (PTKs) are involved in cytokine induction in response to lipopolysaccharides (LPS; TLR4 ligand) in macrophages, the PTK-mediated signal transduction pathway has yet to be analyzed in detail. Here, we carried out a comprehensive and quantitative dynamic tyrosine phosphoproteomic analysis on the TLR4-mediated host defense system in RAW264.7 macrophages using stable isotope labeling by amino acids in cell culture (SILAC). We determined the temporal profiles of 25 proteins based on SILAC-encoded peptide(s). Of these, we focused on the tyrosine phosphorylation of B-cell adaptor for phosphatidylinositol 3-kinase (BCAP) because the function of BCAP remains unknown in TLR signaling in macrophages. Furthermore, Bcap has two distinct transcripts, a full-length (Bcap-{sub L}) and an alternatively initiated or spliced (Bcap-{sub S}) mRNA, and little is known about the differential functions of the BCAP-{sub L} and BCAP-{sub S} proteins. Our study showed, for the first time, that RNAi-mediated selective depletion of BCAP-{sub L} enhanced IL-6 and IL-10 production but not TNF-{alpha} production in TLR ligand-stimulated macrophages. We propose that BCAP-{sub L} (but not BCAP-{sub S}) is a negative regulator of the TLR-mediated host defense system in macrophages.

  5. Regulation of bacteriocin production in Lactobacillus plantarum depends on a conserved promoter arrangement with consensus binding sequence

    NARCIS (Netherlands)

    Risoen, PA; Johnsborg, O; Diep, DB; Hamoen, L; Venema, G; Nes, IF

    Bacteriocin production in Lactobacillus plantarum? C11 is regulated by a three-component signal transduction system comprising a peptide pheromone (PlnA), a histidine protein kinase (PlnB), and two homologous response regulators (RRs; PlnC and PlnD). Both RRs are DNA-binding proteins that bind to

  6. 75 FR 79320 - Animal Drugs, Feeds, and Related Products; Regulation of Carcinogenic Compounds in Food-Producing...

    Science.gov (United States)

    2010-12-20

    ... HUMAN SERVICES Food and Drug Administration 21 CFR Part 500 Animal Drugs, Feeds, and Related Products; Regulation of Carcinogenic Compounds in Food-Producing Animals AGENCY: Food and Drug Administration, HHS... regulations regarding compounds of carcinogenic concern used in food-producing animals. Specifically, the...

  7. Typical investigational medicinal products follow relatively uniform regulations in 10 European Clinical Research Infrastructures Network (ECRIN) countries

    DEFF Research Database (Denmark)

    Gluud, Christian; Kubiak, Christine; Whitfield, Kate

    2012-01-01

    In order to facilitate multinational clinical research, regulatory requirements need to become international and harmonised. The EU introduced the Directive 2001/20/EC in 2004, regulating investigational medicinal products in Europe.......In order to facilitate multinational clinical research, regulatory requirements need to become international and harmonised. The EU introduced the Directive 2001/20/EC in 2004, regulating investigational medicinal products in Europe....

  8. Fis is a global regulator critical for modulation of virulence factor production and pathogenicity of Dickeya zeae

    OpenAIRE

    Lv, Mingfa; Chen, Yufan; Liao, Lisheng; Liang, Zhibin; Shi, Zurong; Tang, Yingxin; Ye, Sixuan; Zhou, Jianuan; Zhang, Lianhui

    2018-01-01

    Dickeya zeae is the causal agent of rice foot rot disease, which has recently become a great threat to rice planting countries and regions. The pathogen produces a family of phytotoxins named zeamines that is critical for bacterial virulence, but little is known about the signaling pathways and regulatory mechanisms that govern zeamine production. In this study, we showed that a conserved transcriptional regulator Fis is involved in the regulation of zeamine production in D. zeae strain EC1. ...

  9. The changes in hazard classification and product notification procedures of the new European CLP and Cosmetics Regulations.

    Science.gov (United States)

    de Groot, Ronald; Brekelmans, Pieter; Herremans, Joke; Meulenbelt, Jan

    2010-01-01

    The United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN-GHS) is developed to harmonize the criteria for hazard communication worldwide. The European Regulation on classification, labeling, and packaging of substances and mixtures [CLP Regulation (European Commission, EC) No 1272/2008] will align the existing European Union (EU) legislation to the UN-GHS. This CLP Regulation entered into force on January 20, 2009, and will, after a transitional period, replace the current rules on classification, labeling, and packaging for supply and use in Europe. Both old and new classifications will exist simultaneously until 2010 for substances and until 2015 for mixtures. The new hazard classification will introduce new health hazard classes and categories, with associated new hazard pictograms, signal words, Hazard (H)-statements, and Precautionary (P)-statements as labeling elements. Furthermore, the CLP Regulation will affect the notification of product information on hazardous products to poisons information centers (PICs). At this moment product notification widely varies in procedures and requirements across EU Member States. Article 45 of the CLP Regulation contains a provision stating that the EC will (by January 20, 2012) review the possibility of harmonizing product notification. The European Association of Poisons Centres and Clinical Toxicologists (EAPCCT) is recognized as an important stakeholder. For cosmetic products, the new Cosmetics Regulation will directly implement a new procedure for electronic cosmetic product notification in all EU Member States. Both the CLP Regulation and the Cosmetics Regulation will develop their own product notification procedure within different time frames. Harmonization of notification procedures for both product groups, especially a common electronic format, would be most effective from a cost-benefit viewpoint and would be welcomed by PICs.

  10. A comparison of US and Norwegian regulation of coumarin in tobacco products.

    Science.gov (United States)

    Givel, M

    2003-12-01

    This paper examines policy processes regarding why the USA and Norway have not regulated coumarin in tobacco. A qualitative analysis of all tobacco industry documents regarding coumarin since the 1950s from the 1998 US Master Settlement Agreement and subsequent legal settlements. Additional data were collected from newspaper reports, general internet search engines, journal articles, scholarly reports, court cases, statutes, regulations, and informal correspondence with tobacco control experts in Norway. An overview, summary, and analysis of all documents related to coumarin. In the USA from 1954 until 1985 when coumarin was reportedly removed from domestic cigarettes, but not from pipe tobacco until 1996, and not at all from imported Indian bidi cigarettes, regulatory efforts were stymied. In Norway, from 1973 to the present, the tobacco industry has never disclosed whether its tobacco products contain coumarin. In both the USA and Norway, the extreme delay and lack of vigorous evidence gathering and significant remedies were caused by tobacco industry assertions that revealing tobacco additives was a violation of trade secrets, and by weak regulatory authority and efforts to regulate coumarin. Vigorous and expeditious regulatory investigations and remedies for harmful additives in tobacco, such as coumarin, can protect the public health. Astute insider and outsider political advocacy by health advocates is required to hold elected officials and civil servants publicly accountable for failing to enact disclosure laws and to engage in effective regulatory efforts.

  11. Four hundred and sixty brands of e-cigarettes and counting: implications for product regulation.

    Science.gov (United States)

    Zhu, Shu-Hong; Sun, Jessica Y; Bonnevie, Erika; Cummins, Sharon E; Gamst, Anthony; Yin, Lu; Lee, Madeleine

    2014-07-01

    E-cigarettes are largely unregulated and internet sales are substantial. This study examines how the online market for e-cigarettes has changed over time: in product design and in marketing messages appearing on websites. Comprehensive internet searches of English-language websites from May-August 2012 and December 2013-January 2014 identified brands, models, flavours, nicotine strengths, ingredients and product claims. Brands were divided into older and newer groups (by the two searches) for comparison. By January 2014 there were 466 brands (each with its own website) and 7764 unique flavours. In the 17 months between the searches, there was a net increase of 10.5 brands and 242 new flavours per month. Older brands were more likely than newer brands to offer cigalikes (86.9% vs. 52.1%, pcigarettes, were good substitutes where smoking was banned and were effective smoking cessation aids. Newer brands offered more flavours per brand (49 vs. 32, pcigarettes. The number of e-cigarette brands is large and has been increasing. Older brands tend to highlight their advantages over conventional cigarettes while newer brands emphasise consumer choice in multiple flavours and product versatility. These results can serve as a benchmark for future research on the impact of upcoming regulations on product design and advertising messages of e-cigarettes. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  12. Comparative studies of oil product regulation in polluted soil for several industrialized countries

    Science.gov (United States)

    Paccassoni, F.; Kalnina, D.; Piga, L.

    2017-10-01

    Oil contaminated sites are the consequence of a long period of industrialization. Oil is a complex mixture including aliphatic and aromatic hydrocarbons, which are known to have negative effects on human health and the environment. Dividing oil products in groups (fractions) of petroleum hydrocarbons that act alike in soil and water, one can better know what happens to them. Being able to understand the behaviour of oil products in soil, it will allow to implement prevention and remediation actions. Interventions on contaminated sites are bound to comply with regulatory limits that each country has set in their own environmental legislation. The different concentration thresholds of oil products in soil for several EU countries and Canada has led to compare: limit values, analytical method, soil characteristics and/or land use. This will allow to evaluate what could be the best regulation approach, assessing if it is better to consider soil matrix in the site or the specific land use or both of them. It will also assess what is the best analytical methodology to be adopted to achieve the pollutant concentrations in the soil in order to have comparable results among different countries, such as: Baltic countries (Latvia, Estonia, Lithuania), Nordic countries (Finland, Sweden, Norway, Denmark), Western countries (Italy and The Netherlands) and Canada, like gaschromatography in the range from C10 - C50. The study presents an overview of environmental regulatory system of several EU countries and Canada and the correlation between different parameters about oil products indicated in each environmental legislation.

  13. Adipocyte lipid chaperone AP2 is a secreted adipokine regulating hepatic glucose production.

    Science.gov (United States)

    Cao, Haiming; Sekiya, Motohiro; Ertunc, Meric Erikci; Burak, M Furkan; Mayers, Jared R; White, Ariel; Inouye, Karen; Rickey, Lisa M; Ercal, Baris C; Furuhashi, Masato; Tuncman, Gürol; Hotamisligil, Gökhan S

    2013-05-07

    Proper control of hepatic glucose production is central to whole-body glucose homeostasis, and its disruption plays a major role in diabetes. Here, we demonstrate that although established as an intracellular lipid chaperone, aP2 is in fact actively secreted from adipocytes to control liver glucose metabolism. Secretion of aP2 from adipocytes is regulated by fasting- and lipolysis-related signals, and circulating aP2 levels are markedly elevated in mouse and human obesity. Recombinant aP2 stimulates glucose production and gluconeogenic activity in primary hepatocytes in vitro and in lean mice in vivo. In contrast, neutralization of secreted aP2 reduces glucose production and corrects the diabetic phenotype of obese mice. Hyperinsulinemic-euglycemic and pancreatic clamp studies upon aP2 administration or neutralization demonstrated actions of aP2 in liver. We conclude that aP2 is an adipokine linking adipocytes to hepatic glucose production and that neutralizing secreted aP2 may represent an effective therapeutic strategy against diabetes. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. REGULATION OF PRODUCTION PERFORMANCE OF CHICORY PLANTS BY FOLIAR APPLICATION OF BIOLOGICALLY ACTIVE SUBSTANCES

    Directory of Open Access Journals (Sweden)

    MAREK KOVÁR

    2012-12-01

    Full Text Available In this study were evaluated both the growth and yield potentials of three chicory (Cichorium intybus var. sativum varieties ('Fredonia Nova', 'Oesia' a 'Maurane' growing in natural agro-ecological conditions from 2006 to 2008. Regulation of the crop productivity by foliar application of biologically active substances (Atonik, Polybor 150, and Biafit Gold was also studied. Evaluation of growth-production performance of chicory was realized as: leaf area index (LAI, photosynthetic potential (LAD, net assimilation rate (NAR, crop rate growth (CGR, relative growth rate (RGR, harvest index (HI, root yield and inulin content in root. During threeyears of experimental seasons, the highest average yield of chicory roots was observed in variety 'Fredonia Nova' (value 2.08 kg m-2, with average LAI reached on value 1.88 and average NAR on value 2.15 g m-2 d-1. The highest average HI was observed in variety 'Oesia' (64.0 % and the lowest in variety 'Maurane' (57.3 %. The highest average inulin root content reached variety 'Fredonia Nova' up to value 205.9 mg g-1 dry weight. In this study we discussed in detail observed interaction between individual growth-production characteristics. Finally we observed that application of biologically active substances (BAS is an important tool for optimalization of growthproduction potential in chicory plant. Our results showed that application of BAS (and mainly of Atonik in all the varieties and experimental years significantly increased the growth-production performance and modified source – sink relationships.

  15. Silibinin regulates gene expression, production and secretion of mucin from cultured airway epithelial cells.

    Science.gov (United States)

    Kim, Kil-Dong; Lee, Hyun Jae; Lim, Seung Pyong; Sikder, Asaduzzaman; Lee, Su Yel; Lee, Choong Jae

    2012-09-01

    We investigated whether silibinin significantly affects gene expression, production and secretion of mucin from cultured airway epithelial cells. Confluent NCI-H292 cells were pretreated with silibinin for 30 min and then stimulated with epidermal growth factor (EGF), phorbol 12-myristate 13-acetate (PMA) or TNF-α for 24 h. The MUC5AC mucin gene expression and mucin protein production were measured by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). The effect of silibinin on TNF-α-induced activation of NF-κB p65 was also examined. Confluent primary rat tracheal surface epithelial (RTSE) cells were pretreated with adenosine triphosphate (ATP) for 5 min and then treated for 30 min in the presence of silibinin to assess the effect on mucin secretion using ELISA. The results were as follows: (i) silibinin inhibited the expression of the MUC5AC mucin gene induced by EGF, PMA or TNF-α from NCI-H292 cells; (ii) silibinin also inhibited the production of MUC5AC mucin protein induced by the same inducers from NCI-H292 cells; (iii) silibinin inhibited the activation of NF-κB p65 by TNF-α in NCI-H292 cells; (iv) silibinin significantly decreased ATP-induced mucin secretion from cultured RTSE cells. This result suggests that silibinin can regulate gene expression, production and secretion of mucin by directly acting on airway epithelial cells. Copyright © 2012 John Wiley & Sons, Ltd.

  16. Environmental Regulations on Waste Electrical Equipment Industry: The Production Chain to the Final Consumer

    Directory of Open Access Journals (Sweden)

    Marilia Tunes Mazon

    2012-06-01

    Full Text Available The relevance of the electronics industry requires its transition to sustainable production patterns, as defined by the European directives WEEE, RoHS, and the National Policy on Solid Waste. In 2010, the group began a national project whose purpose is to support sustainability with a focus on the triple bottom line Brazilian electronics industry. The implementation of these proposals began with the implementation of a pilot project with nine companies of electromedical equipment, chosen because they are technology intensive, and require exporters suit environmental regulations. This paper aims to present the main results obtained in the studies with these nine companies in the period Apr/2011 Mar/2012 regarding the suitability of its products, processes, management systems and supply chains. The study was divided into three stages: an assessment to identify general aspects of each organization, a thorough assessment to understand the characteristics of each organization ahead of the general characteristics of the group analyzed and a detailed diagnosis, performed individually during visits in companies. The main findings were that the waste management and reverse logistics associated are still incipient, the products still have dangerous substances and supply chain is still very poorly prepared for the challenges. The level of employment of employees and costs associated with the adequacy of its products and processes, constitute the two biggest problems for companies fit the relevant environmental legislation.

  17. Statins Activate Human PPAR Promoter and Increase PPAR mRNA Expression and Activation in HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Makoto Seo

    2008-01-01

    Full Text Available Statins increase peroxisome proliferator-activated receptor (PPAR mRNA expression, but the mechanism of this increased PPAR production remains elusive. To examine the regulation of PPAR production, we examined the effect of 7 statins (atorvastatin, cerivastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin on human PPAR promoter activity, mRNA expression, nuclear protein levels, and transcriptional activity. The main results are as follows. (1 Majority of statins enhanced PPAR promoter activity in a dose-dependent manner in HepG2 cells transfected with the human PPAR promoter. This enhancement may be mediated by statin-induced HNF-4. (2 PPAR mRNA expression was increased by statin treatment. (3 The PPAR levels in nuclear fractions were increased by statin treatment. (4 Simvastatin, pravastatin, and cerivastatin markedly enhanced transcriptional activity in 293T cells cotransfected with acyl-coenzyme A oxidase promoter and PPAR/RXR expression vectors. In summary, these data demonstrate that PPAR production and activation are upregulated through the PPAR promoter activity by statin treatment.

  18. Economic Analysis of Legal Regulations Regarding Liability for Loss Resulting from a Dangerous Product

    Directory of Open Access Journals (Sweden)

    Joanna Kuźmicka-Sulikowska

    2013-06-01

    Full Text Available The article considers fundamental issues associated with economic analysis of legal regulations pertaining to liability for damage caused by a dangerous product. The discussion is conducted from the perspective of a lawyer analysing the conclusions stemming from economic analyses of the aforementioned liability by individual authors, which constitutes a base for subsequent observations. The relationships between the various potential configurations of manufacturer liability are discussed, in particular a situation where such liability is not stipulated, as well as where there is the introduction of liability based on the risk or fault principles and the ensuing motivation for maintaining an appropriate level of carefulness. Attention is focused on the search for an economically efficient solution. An analysis of current regulation is performed, as well as de lege ferenda conclusions grounded in economic analysis of law with reference to the shape of the regulatory liability regime for damage caused by a dangerous product. Many reservations are also listed, including ones concerning the fact that it does not seem that the legislative process should be guided only by conclusions stemming from economic analysis of law (especially considering the differences of opinion in that area, which does, however, unquestionably constitute a useful tool in such matters.

  19. The second transferrin receptor regulates red blood cell production in mice.

    Science.gov (United States)

    Nai, Antonella; Lidonnici, Maria Rosa; Rausa, Marco; Mandelli, Giacomo; Pagani, Alessia; Silvestri, Laura; Ferrari, Giuliana; Camaschella, Clara

    2015-02-12

    Transferrin receptor 2 (TFR2) contributes to hepcidin regulation in the liver and associates with erythropoietin receptor in erythroid cells. Nevertheless, TFR2 mutations cause iron overload (hemochromatosis type 3) without overt erythroid abnormalities. To clarify TFR2 erythroid function, we generated a mouse lacking Tfr2 exclusively in the bone marrow (Tfr2(BMKO)). Tfr2(BMKO) mice have normal iron parameters, reduced hepcidin levels, higher hemoglobin and red blood cell counts, and lower mean corpuscular volume than normal control mice, a phenotype that becomes more evident in iron deficiency. In Tfr2(BMKO) mice, the proportion of nucleated erythroid cells in the bone marrow is higher and the apoptosis lower than in controls, irrespective of comparable erythropoietin levels. Induction of moderate iron deficiency increases erythroblasts number, reduces apoptosis, and enhances erythropoietin (Epo) levels in controls, but not in Tfr2(BMKO) mice. Epo-target genes such as Bcl-xL and Epor are highly expressed in the spleen and in isolated erythroblasts from Tfr2(BMKO) mice. Low hepcidin expression in Tfr2(BMKO) is accounted for by erythroid expansion and production of the erythroid regulator erythroferrone. We suggest that Tfr2 is a component of a novel iron-sensing mechanism that adjusts erythrocyte production according to iron availability, likely by modulating the erythroblast Epo sensitivity. © 2015 by The American Society of Hematology.

  20. Natural Compounds as Regulators of NLRP3 Inflammasome-Mediated IL-1β Production

    Science.gov (United States)

    2016-01-01

    IL-1β is one of the main proinflammatory cytokines that regulates a broad range of immune responses and also participates in several physiological processes. The canonical production of IL-1β requires multiprotein complexes called inflammasomes. One of the most intensively studied inflammasome complexes is the NLRP3 inflammasome. Its activation requires two signals: one signal “primes” the cells and induces the expression of NLRP3 and pro-IL-1β, while the other signal leads to the assembly and activation of the complex. Several stimuli were reported to function as the second signal including reactive oxygen species, lysosomal rupture, or cytosolic ion perturbation. Despite very intensive studies, the precise function and regulation of the NLRP3 inflammasome are still not clear. However, many chronic inflammatory diseases are related to the overproduction of IL-1β that is mediated via the NLRP3 inflammasome. In this review, we aimed to provide an overview of studies that demonstrated the effect of plant-derived natural compounds on NLRP3 inflammasome-mediated IL-1β production. Although many of these studies lack the mechanistic explanation of their action, these compounds may be considered as complementary supplements in the treatment of chronic inflammatory diseases, consumed as preventive agents, and may also be considered as molecular tools to study NLRP3 function. PMID:27672241

  1. Indirect Regulation of Endogenous Glucose Production by Insulin: The Single Gateway Hypothesis Revisited.

    Science.gov (United States)

    Bergman, Richard N; Iyer, Malini S

    2017-07-01

    On the basis of studies that investigated the intraportal versus systemic insulin infusion and transendothelial transport of insulin, we proposed the "single gateway hypothesis," which supposes an indirect regulation of hepatic glucose production by insulin; the rate-limiting transport of insulin across the adipose tissue capillaries is responsible for the slow suppression of free fatty acids (FFAs), which in turn is responsible for delayed suppression of hepatic endogenous glucose production (EGP) during insulin infusion. Preventing the fall in plasma FFAs during insulin infusion either by administering intralipids or by inhibiting adipose tissue lipolysis led to failure in EGP suppression, thus supporting our hypothesis. More recently, mice lacking hepatic Foxo1 in addition to Akt1 and Akt2 (L-AktFoxo1TKO), all required for insulin signaling, surprisingly showed normal glycemia. Inhibiting the fall of plasma FFAs in these mice prevented the suppression of EGP during a clamp, reaffirming that the site of insulin action to control EGP is extrahepatic. Measuring whole-body turnover rates of glucose and FFAs in L-AktFoxo1TKO mice also confirmed that hepatic EGP was regulated by insulin-mediated control of FFAs. The knockout mouse model in combination with sophisticated molecular techniques confirmed our physiological findings and the single gateway hypothesis. © 2017 by the American Diabetes Association.

  2. S-(–)equol production is developmentally regulated and related to early diet composition

    Science.gov (United States)

    Brown, Nadine M.; Galandi, Stephanie L.; Summer, Suzanne S.; Zhao, Xueheng; Heubi, James E.; King, Eileen C.; Setchell, Kenneth D.R.

    2016-01-01

    S-(−)7-hydroxy-3-(4′-hydroxyphenyl)-chroman, or S-(−)equol, a biologically active intestinally derived bacterial metabolite of the soy isoflavones daidzin/daidzein, is not produced in neonatal life. Because its synthesis is dependent on equol-producing bacteria, we hypothesized that early nutrition may influence equol production. This prospective 2.5-year study determined the frequency of S-(−)equol production in healthy infants (n = 90) fed breast milk, soy infant formula, or cow’s milk formula in their first year. Urinary S-(−)equol and daidzein were quantified by mass spectrometry after a standardized 3.5-day soy isoflavone challenge. Infants were tested at 6, 9, 12, 18, 24, and 36 months of age, and 3-day diet records were obtained at each visit to explore the effect of early and postweaning (>12 months) macronutrient and micronutrient dietary composition and S-(−)equol production. Use of antibiotics was also recorded. At age 6 months, none of the breast-fed infants produced S-(−)equol, whereas 3.8% and 6.0%, respectively, of soy and cow’s milk formula–fed infants were equol producers. By age 3 years, 50% of the formula-fed infants were equol producers, compared with 25% of breast-fed infants. Use of antibiotics was prevalent among infants and may have impacted the stability of S-(−)equol production. No significant differences among the groups were observed in postweaning dietary intakes of total energy, carbohydrate, fiber, protein, fat, saturated fatty acids, or polyunsaturated fatty acids and the propensity to make S-(−)equol. In conclusion, S-(−)equol production is developmentally regulated and initially related to diet composition with the proportion of equol producers increasing over the first 3 years of life, with a trend for formula feeding favoring S-(−)equol production. PMID:24916553

  3. S-(-)equol production is developmentally regulated and related to early diet composition.

    Science.gov (United States)

    Brown, Nadine M; Galandi, Stephanie L; Summer, Suzanne S; Zhao, Xueheng; Heubi, James E; King, Eileen C; Setchell, Kenneth D R

    2014-05-01

    S-(-)7-hydroxy-3-(4'-hydroxyphenyl)-chroman, or S-(-)equol, a biologically active intestinally derived bacterial metabolite of the soy isoflavones daidzin/daidzein, is not produced in neonatal life. Because its synthesis is dependent on equol-producing bacteria, we hypothesized that early nutrition may influence equol production. This prospective 2.5-year study determined the frequency of S-(-)equol production in healthy infants (n = 90) fed breast milk, soy infant formula, or cow's milk formula in their first year. Urinary S-(-)equol and daidzein were quantified by mass spectrometry after a standardized 3.5-day soy isoflavone challenge. Infants were tested at 6, 9, 12, 18, 24, and 36 months of age, and 3-day diet records were obtained at each visit to explore the effect of early and postweaning (>12 months) macronutrient and micronutrient dietary composition and S-(-)equol production. Use of antibiotics was also recorded. At age 6 months, none of the breast-fed infants produced S-(-)equol, whereas 3.8% and 6.0%, respectively, of soy and cow's milk formula-fed infants were equol producers. By age 3 years, 50% of the formula-fed infants were equol producers, compared with 25% of breast-fed infants. Use of antibiotics was prevalent among infants and may have impacted the stability of S-(-)equol production. No significant differences among the groups were observed in postweaning dietary intakes of total energy, carbohydrate, fiber, protein, fat, saturated fatty acids, or polyunsaturated fatty acids and the propensity to make S-(-)equol. In conclusion, S-(-)equol production is developmentally regulated and initially related to diet composition with the proportion of equol producers increasing over the first 3 years of life, with a trend for formula feeding favoring S-(-)equol production. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. ApoE production in human monocytes and its regulation by inflammatory cytokines.

    Directory of Open Access Journals (Sweden)

    Sten Braesch-Andersen

    Full Text Available The apoE production by tissue macrophages is crucial for the prevention of atherosclerosis and the aim of this study was to further elucidate how this apolipoprotein is regulated by cytokines present during inflammation. Here we studied apoE production in peripheral blood mononuclear cells (PBMC and analysis was made with a newly developed apoE ELISpot assay. In PBMC, apoE secretion was restricted to monocytes with classical (CD14(++CD16(- and intermediate (CD14(+CD16(+ monocytes being the main producers. As earlier described for macrophages, production was strongly upregulated by TGF-β and downregulated by bacterial lipopolysaccharide (LPS and the inflammatory cytokines IFN-γ, TNF-α and IL-1β. We could here show that a similar down-regulatory effect was also observed with the type I interferon, IFN-α, while IL-6, often regarded as one of the more prominent inflammatory cytokines, did not affect TGF-β-induced apoE production. The TNF-α inhibitor Enbrel could partly block the down-regulatory effect of IFN-γ, IFN-α and IL-1β, indicating that inhibition of apoE by these cytokines may be dependent on or synergize with TNF-α. Other cytokines tested, IL-2, IL-4, IL-12, IL-13, IL-17A and IL-23, had no inhibitory effect on apoE production. In contrast to the effect on monocytes, apoE production by primary hepatocytes and the hepatoma cell line HepG2 was more or less unaffected by treatment with cytokines or LPS.

  5. Regulation of schistosome egg production by HMG CoA reductase

    Energy Technology Data Exchange (ETDEWEB)

    VandeWaa, E.A.; Bennett, J.L.

    1986-03-05

    Hydroxymethylglutaryl coenzyme A reductase (HMG CoA reductase) catalyzes the conversion of HMG CoA to mevalonate in the synthesis of steroids, isoprenoids and terpenes. Mevinolin, an inhibitor of this enzyme, decreased egg production in Schistosoma mansoni during in vitro incubations. This was associated with a reduction in the incorporation of /sup 14/C-acetate into polyisoprenoids and a reduction in the formation of a lipid-linked oligosaccharide. In vivo, mevinolin in daily doses of 50 mg/kg (p.o., from days 30-48 post-infection) caused no change in gross liver pathology in S. mansoni infected mice. However, when parasites exposed to mevinolin or its vehicle in vivo were cultured in vitro, worms from mevinolin-treated mice produced six times more eggs than control parasites. When infected mice were dosed with 250 mg/kg mevinolin daily (p.o., from days 35-45 post-infection), liver pathology was reduced in comparison to control mice. Thus, during in vivo exposure to a high dose of the drug egg production is decreased, while at a lower dose it appears unaffected until the parasites are cultured in a drug-free in vitro system wherein egg production is stimulated to extraordinarily high levels. It may be that at low doses mevinolin, by inhibiting the enzyme, is blocking the formation of a product (such as an isoprenoid) which normally acts to down-regulate enzyme synthesis, resulting in enzyme induction. Induction of HMG CoA reductase is then expressed as increased egg production when the worms are removed from the drug. These data suggest that HMG CoA reductase plays a role in schistosome egg production.

  6. The Impact of Agricultural Products Insurance on Using Technical-Healthy Regulations of Production: A Case Study of Industrial Livestock and Apple Farms

    Directory of Open Access Journals (Sweden)

    A. Falsafian

    2011-06-01

    Full Text Available Agricultural insurance is considered as a support policy that reduces negative effects of risk in agricultural farms. In addition, it can improve managerial behavior of insured and technical-healthy regulations of production by using some special tools. This study investigated the impact ofinsurance of agricultural products insurance on using technical-healthy regulations of production in industrial livestock farms at Khorasan province and apple farms at Damavand County. To this end, the required data were survyed from the farms; then it was statistically analyzed. Results indicated that the insurance has exactly significant influence on using any of technical-healthy regulations of production in industrial livestock farms; whereas the insurance does not have any significant influence in the apple farms. In addition, it was also found out that the greatest influence of insurance was in industrial livestock and apple farms that had less than 200 animals and 3 hectares farms, respectively.

  7. IgG-derived Fc down-regulates virus-induced plasmacytoid dendritic cell (pDC) IFNalpha production.

    Science.gov (United States)

    Green, Daniel S; Lum, Tom; Green, Jon A

    2004-06-07

    Interferon alpha (IFNalpha) produced primarily by plasmacytoid dendritic cells (pDC) is a potent component of the anti-viral innate immune response, and modulates adaptive immunity. Primary control of IFNalpha production occurs at a cellular level and is highly dependent upon regulatory factors and their products. Recent studies have identified up-regulation of IFNalpha production mediated by the adaptive immune response in the form of immune specific IgG. These studies establish a role for the external control of IFNalpha production. The current work demonstrates that the Fc portion of IgG is a potent inhibitor of IFNalpha produced by pDCs in response to HIV, HSV, and VSV. Fc down-regulation occurs after IFNalpha production can be detected by bioassay, and suggests the existence of late regulatory events in the control of IFNalpha production. Down-regulation of IFNalpha is not caused by Fc-induced necrosis, apoptosis or neutralization of IFNalpha activity. Demonstration of Fc-mediated down-regulation of IFNalpha provides additional evidence of the role of IgG in the regulation of IFNalpha production. Elsevier Ltd.

  8. Endogenous Production of Extracellular Adenosine by Trabecular Meshwork Cells: Potential Role in Outflow Regulation

    Science.gov (United States)

    Wu, Jing; Li, Guorong; Luna, Coralia; Spasojevic, Ivan; Epstein, David L.; Gonzalez, Pedro

    2012-01-01

    Purpose. To investigate the mechanisms for endogenous production of extracellular adenosine in trabecular meshwork (TM) cells and evaluate its physiological relevance to the regulation of aqueous humor outflow facility. Methods. Extra-cellular levels of adenosine monophosphate (AMP) and adenosine in porcine trabecular meshwork (PTM) cells treated with adenosine triphosphate (ATP), AMP, cAMP or forskolin with or without specific inhibitors of phosphodiesterases (IBMX) and CD73 (AMPCP) were determined by high-pressure liquid chromatography fluorometry. Extracellular adenosine was also evaluated in cell cultures subjected to cyclic mechanical stress (CMS) (20% stretching; 1 Hz) and after disruption of lipid rafts with methyl-β-cyclodextrin. Expression of CD39 and CD73 in porcine TM cells and tissue were examined by Q-PCR and Western blot. The effect of inhibition of CD73 on outflow facility was evaluated in perfused living mouse eyes. Results. PTM cells generated extracellular adenosine from extracellular ATP and AMP but not from extracellular cAMP. Increased intracellular cAMP mediated by forskolin led to a significant increase in extracellular adenosine production that was not prevented by IBMX. Inhibition of CD73 resulted, in all cases, in a significant decrease in extracellular adenosine. CMS induced a significant activation of extracellular adenosine production. Inhibition of CD73 activity with AMPCP in living mouse eyes resulted in a significant decrease in outflow facility. Conclusions. These results support the concept that the extracellular adenosine pathway might play an important role in the homeostatic regulation of outflow resistance in the TM, and suggest a novel mechanism by which pathologic alteration of the TM, such as increased tissue rigidity, could lead to abnormal elevation of IOP in glaucoma. PMID:22997289

  9. Influence of pH Regulation Mode in Glucose Fermentation on Product Selection and Process Stability.

    Science.gov (United States)

    Mohd-Zaki, Zuhaida; Bastidas-Oyanedel, Juan R; Lu, Yang; Hoelzle, Robert; Pratt, Steven; Slater, Fran R; Batstone, Damien J

    2016-01-04

    Mixed culture anaerobic fermentation generates a wide range of products from simple sugars, and is potentially an effective process for producing renewable commodity chemicals. However it is difficult to predict product spectrum, and to control the process. One of the key control handles is pH, but the response is commonly dependent on culture history. In this work, we assess the impact of pH regulation mode on the product spectrum. Two regulation modes were applied: in the first, pH was adjusted from 4.5 to 8.5 in progressive steps of 0.5 and in the second, covered the same pH range, but the pH was reset to 5.5 before each change. Acetate, butyrate, and ethanol were produced throughout all pH ranges, but there was a shift from butyrate at pH pH > 6.5, as well as a strong and consistent shift from hydrogen to formate as pH increased. Microbial analysis indicated that progressive pH resulted in dominance by Klebsiella, while reset pH resulted in a bias towards Clostridium spp., particularly at low pH, with higher variance in community between different pH levels. Reset pH was more responsive to changes in pH, and analysis of Gibbs free energy indicated that the reset pH experiments operated closer to thermodynamic equilibrium, particularly with respect to the formate/hydrogen balance. This may indicate that periodically resetting pH conforms better to thermodynamic expectations.

  10. Influence of pH Regulation Mode in Glucose Fermentation on Product Selection and Process Stability

    Directory of Open Access Journals (Sweden)

    Zuhaida Mohd-Zaki

    2016-01-01

    Full Text Available Mixed culture anaerobic fermentation generates a wide range of products from simple sugars, and is potentially an effective process for producing renewable commodity chemicals. However it is difficult to predict product spectrum, and to control the process. One of the key control handles is pH, but the response is commonly dependent on culture history. In this work, we assess the impact of pH regulation mode on the product spectrum. Two regulation modes were applied: in the first, pH was adjusted from 4.5 to 8.5 in progressive steps of 0.5 and in the second, covered the same pH range, but the pH was reset to 5.5 before each change. Acetate, butyrate, and ethanol were produced throughout all pH ranges, but there was a shift from butyrate at pH < 6.5 to ethanol at pH > 6.5, as well as a strong and consistent shift from hydrogen to formate as pH increased. Microbial analysis indicated that progressive pH resulted in dominance by Klebsiella, while reset pH resulted in a bias towards Clostridium spp., particularly at low pH, with higher variance in community between different pH levels. Reset pH was more responsive to changes in pH, and analysis of Gibbs free energy indicated that the reset pH experiments operated closer to thermodynamic equilibrium, particularly with respect to the formate/hydrogen balance. This may indicate that periodically resetting pH conforms better to thermodynamic expectations.

  11. MCPIP-1, alias Regnase-1 controls epithelial inflammation by post-transcriptional regulation of IL-8 production

    Science.gov (United States)

    Dobosz, E.; Wilamowski, M.; Lech, M.; Bugara, B.; Jura, J.; Potempa, J.; Koziel, J.

    2016-01-01

    Pattern recognition receptors are critical for the detection of invading microorganisms. They activate multiple pathways that lead to the induction of pro-inflammatory responses and pathogen clearance. The intensity and duration of this immune reaction must be tightly controlled spatially and temporally in every tissue by different negative regulators. We hypothesized that monocyte chemoattractant protein-1–induced protein-1 (MCPIP-1) might play a role in maintaining immune homeostasis in the epithelium both under physiological conditions and upon bacterial infection. To this end, we examined the distribution of MCPIP-1 transcript and protein in various tissues. The MCPIP-1 protein level was higher in epithelial cells than in myeloid cells. MCPIP-1 exerted RNase activity towards the IL-8 transcript and the life-span of IL-8 was determined by the presence of the stem-loops/hairpin (SL) structures at the 3′ UTR region of IL-8 mRNA. Moreover, using fully active, purified recombinant MCPIP-1 protein, we elucidated the mechanism by which MCPIP-1 controls the IL-8 mRNA level. In conclusion, we uncovered a novel IL-8–dependent mechanism via which MCPIP-1 maintains epithelial homeostasis. This study reveals for the first time that MCPIP-1 plays a crucial anti-inflammatory role not only in myeloid cells but also in epithelial cells. PMID:27513529

  12. Role of opioid peptides in the regulation of cytokine production by murine CD4+ T cells.

    Science.gov (United States)

    van den Bergh, P; Dobber, R; Ramlal, S; Rozing, J; Nagelkerken, L

    1994-03-01

    The presence of the opioid peptides alpha- and beta-endorphin (-End) but not methionine enkephalin (Met-enk) in in vitro cultures of purified CD4+ T cells, stimulated with concanavalin A in the presence of irradiated spleen cells, resulted in a threefold stimulation of IL-2, IL-4, and IFN-gamma production. The stimulating effect was dependent on the concentration of the peptides and reached optimal values in the dose range from 10(-12) to 10(-10) M. Similar results were obtained when purified CD4+ T cells were stimulated with immobilized anti-CD3, indicating a direct effect of opioid peptides on CD4+ T cells. Moreover, in this system a twofold enhancement of IL-6, but not IL-1, secretion was observed. These stimulatory effects were not mediated through opioid receptors since the peptide fragment beta-End6-31 that lacks the N-terminal opioid receptor binding part was still stimulatory. This is in agreement with our finding that beta-End did not affect cAMP, as described for the triggering of classical opioid receptors. Experiments undertaken to reveal the mechanism of action of opioid peptides suggest an overall enhancement of lymphokine production: (1) enhancement of IL-4 production occurred also in the presence of excess IL-2; and (2) neither IL-1 receptor-antagonizing protein nor anti-IL-6 were capable to abrogate the stimulatory effect on IL-2 and IL-4 production. Finally, the presence and activity of opioid receptors in cultures of CD4+ T cells were substantiated by the fact that the opioid receptor antagonist naloxone by itself enhanced cytokine synthesis, which points to the endogenous production by lymphocytes of down-regulating opioid peptides.

  13. Regulation of xanthine dehydrogensase gene expression and uric acid production in human airway epithelial cells.

    Science.gov (United States)

    Huff, Ryan D; Hsu, Alan C-Y; Nichol, Kristy S; Jones, Bernadette; Knight, Darryl A; Wark, Peter A B; Hansbro, Philip M; Hirota, Jeremy A

    2017-01-01

    The airway epithelium is a physical and immunological barrier that protects the pulmonary system from inhaled environmental insults. Uric acid has been detected in the respiratory tract and can function as an antioxidant or damage associated molecular pattern. We have demonstrated that human airway epithelial cells are a source of uric acid. Our hypothesis is that uric acid production by airway epithelial cells is induced by environmental stimuli associated with chronic respiratory diseases. We therefore examined how airway epithelial cells regulate uric acid production. Allergen and cigarette smoke mouse models were performed using house dust mite (HDM) and cigarette smoke exposure, respectively, with outcome measurements of lung uric acid levels. Primary human airway epithelial cells isolated from clinically diagnosed patients with asthma and chronic obstructive pulmonary disease (COPD) were grown in submerged cultures and compared to age-matched healthy controls for uric acid release. HBEC-6KT cells, a human airway epithelial cell line, were grown under submerged monolayer conditions for mechanistic and gene expression studies. HDM, but not cigarette smoke exposure, stimulated uric acid production in vivo and in vitro. Primary human airway epithelial cells from asthma, but not COPD patients, displayed elevated levels of extracellular uric acid in culture. In HBEC-6KT, production of uric acid was sensitive to the xanthine dehydrogenase (XDH) inhibitor, allopurinol, and the ATP Binding Cassette C4 (ABCC4) inhibitor, MK-571. Lastly, the pro-inflammatory cytokine combination of TNF-α and IFN-γ elevated extracellular uric acid levels and XDH gene expression in HBEC-6KT cells. Our results suggest that the active production of uric acid from human airway epithelial cells may be intrinsically altered in asthma and be further induced by pro-inflammatory cytokines.

  14. Regulation of xanthine dehydrogensase gene expression and uric acid production in human airway epithelial cells.

    Directory of Open Access Journals (Sweden)

    Ryan D Huff

    Full Text Available The airway epithelium is a physical and immunological barrier that protects the pulmonary system from inhaled environmental insults. Uric acid has been detected in the respiratory tract and can function as an antioxidant or damage associated molecular pattern. We have demonstrated that human airway epithelial cells are a source of uric acid. Our hypothesis is that uric acid production by airway epithelial cells is induced by environmental stimuli associated with chronic respiratory diseases. We therefore examined how airway epithelial cells regulate uric acid production.Allergen and cigarette smoke mouse models were performed using house dust mite (HDM and cigarette smoke exposure, respectively, with outcome measurements of lung uric acid levels. Primary human airway epithelial cells isolated from clinically diagnosed patients with asthma and chronic obstructive pulmonary disease (COPD were grown in submerged cultures and compared to age-matched healthy controls for uric acid release. HBEC-6KT cells, a human airway epithelial cell line, were grown under submerged monolayer conditions for mechanistic and gene expression studies.HDM, but not cigarette smoke exposure, stimulated uric acid production in vivo and in vitro. Primary human airway epithelial cells from asthma, but not COPD patients, displayed elevated levels of extracellular uric acid in culture. In HBEC-6KT, production of uric acid was sensitive to the xanthine dehydrogenase (XDH inhibitor, allopurinol, and the ATP Binding Cassette C4 (ABCC4 inhibitor, MK-571. Lastly, the pro-inflammatory cytokine combination of TNF-α and IFN-γ elevated extracellular uric acid levels and XDH gene expression in HBEC-6KT cells.Our results suggest that the active production of uric acid from human airway epithelial cells may be intrinsically altered in asthma and be further induced by pro-inflammatory cytokines.

  15. Regulation of xanthine dehydrogensase gene expression and uric acid production in human airway epithelial cells

    Science.gov (United States)

    Huff, Ryan D.; Hsu, Alan C-Y.; Nichol, Kristy S.; Jones, Bernadette; Knight, Darryl A.; Wark, Peter A. B.; Hansbro, Philip M.

    2017-01-01

    Introduction The airway epithelium is a physical and immunological barrier that protects the pulmonary system from inhaled environmental insults. Uric acid has been detected in the respiratory tract and can function as an antioxidant or damage associated molecular pattern. We have demonstrated that human airway epithelial cells are a source of uric acid. Our hypothesis is that uric acid production by airway epithelial cells is induced by environmental stimuli associated with chronic respiratory diseases. We therefore examined how airway epithelial cells regulate uric acid production. Materials and methods Allergen and cigarette smoke mouse models were performed using house dust mite (HDM) and cigarette smoke exposure, respectively, with outcome measurements of lung uric acid levels. Primary human airway epithelial cells isolated from clinically diagnosed patients with asthma and chronic obstructive pulmonary disease (COPD) were grown in submerged cultures and compared to age-matched healthy controls for uric acid release. HBEC-6KT cells, a human airway epithelial cell line, were grown under submerged monolayer conditions for mechanistic and gene expression studies. Results HDM, but not cigarette smoke exposure, stimulated uric acid production in vivo and in vitro. Primary human airway epithelial cells from asthma, but not COPD patients, displayed elevated levels of extracellular uric acid in culture. In HBEC-6KT, production of uric acid was sensitive to the xanthine dehydrogenase (XDH) inhibitor, allopurinol, and the ATP Binding Cassette C4 (ABCC4) inhibitor, MK-571. Lastly, the pro-inflammatory cytokine combination of TNF-α and IFN-γ elevated extracellular uric acid levels and XDH gene expression in HBEC-6KT cells. Conclusions Our results suggest that the active production of uric acid from human airway epithelial cells may be intrinsically altered in asthma and be further induced by pro-inflammatory cytokines. PMID:28863172

  16. Psidium guajava extract inhibits thymus and activation-regulated chemokine (TARC/CCL17) production in human keratinocytes by inducing heme oxygenase-1 and blocking NF-κB and STAT1 activation.

    Science.gov (United States)

    Han, Eun Hee; Hwang, Yong Pil; Choi, Jae Ho; Yang, Ji Hye; Seo, Jong Kwon; Chung, Young Chul; Jeong, Hye Gwang

    2011-09-01

    Psidium guajava (P. guajava) is a food and medicinal plant with antioxidant, anti-inflammatory, and anti-allergic activities that support its traditional uses. The aim of this study was to determine the effects of P. guajava ethyl acetate extract (PGEA) on atopic dermatitis and to investigate the possible mechanisms by which PGEA inhibits cytokine-induced Th2 chemokine expression in HaCaT human keratinocyte cells. We found that PGEA suppressed the IFN-γ/TNF-α-co-induced production of thymus and activation-regulated chemokine (TARC) protein and mRNA in HaCaT cells. Additionally, PGEA inhibited the TNF-α/IFN-γ-co-induced activation of NF-κB and STAT1 and increased the expression of heme oxygenase-1 (HO-1) protein and mRNA. HO-1 inhibitor enhanced the suppressive effects of PGEA on TNF-α/IFN-γ-co-induced TARC production and gene expression. Collectively, these data demonstrate that PGEA inhibits chemokine expression in keratinocytes by inducing HO-1 expression and it suggests a possible therapeutic application in atopic dermatitis and other inflammatory skin diseases. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. Role of glycolytic intermediate in regulation: Improving lycopene production in Escherichia coli by engineering metabolic control

    Energy Technology Data Exchange (ETDEWEB)

    Farmer, W.R.; Liao, J.C.

    2001-06-01

    Metabolic engineering in the postgenomic era is expected to benefit from a full understanding of the biosynthetic capability of microorganisms as a result of the progress being made in bioinformatics and functional genomics. The immediate advantage of such information is to allow the rational design of novel pathways and the elimination of native reactions that are detrimental or unnecessary for the desired purpose. However, with the ability to manipulate metabolic pathways becoming more effective, metabolic engineering will need to face a new challenge: the reengineering of the regulatory hierarchy that controls gene expression in those pathways. In addition to constructing the genetic composition of a metabolic pathway, they propose that it will become just as important to consider the dynamics of pathways gene expression. It has been widely observed that high-level induction of a recombinant protein or pathway leads to growth retardation and reduced metabolic activity. These phenotypic characteristics result from the fact that the constant demands of production placed upon the cell interfere with its changing requirements for growth. They believe that this common situation in metabolic engineering can be alleviated by designing a dynamic controller that is able to sense the metabolic state of the cell and regulate the expression of the recombinant pathway accordingly. This approach, which is termed metabolic control engineering, involves redesigning the native regulatory circuits and applying them to the recombinant pathway. The general goal of such an effort will be to control the flux to the recombinant pathway adaptively according to the cell's metabolic state. The dynamically controlled recombinant pathway can potentially lead to enhanced production, minimized growth retardation, and reduced toxic by-product formation. The regulation of gene expression in response to the physiological state is also essential to the success of gene therapy. Here they

  18. The impact of lignin down regulation on alfalfa yield, chemical composition and in vitro gas production.

    Science.gov (United States)

    Getachew, G; Laca, E A; Putnam, D H; Witte, D; McCaslin, M; Ortega, K P; DePeters, E J

    2018-02-06

    Lignin is a complex, phenolic polymer found in plant cell walls that is essential for mechanical support, water and mineral transport, and defense in vascular plants. Over 10 different enzymes play a role in the synthesis of lignin in plants. Suppression of any one enzyme or combinations of these enzymes may change the concentration and composition of lignin in the genetically transformed plants. Two lines of alfalfa that were down regulated for caffeoyl-CoA O-methyltransferase (CCOMT) were used to assess the impact of lignin down-regulation on chemical composition and fermentation rate and extent using an in vitro gas production technique. A total of 64 samples consisting two reduced lignin (RL) and two controls (CL)), four field replicates, two cutting intervals (CI) (28 and 35 days) and two Cuts (Cut -1 and Cut-3) were used. No differences were detected in yield, CP, NDF, and ADF between the lines when harvested at the 28-day CI. The ADL concentration in RL alfalfa lines was significantly (p<0.001) lower than in the CL. In alfalfa harvested at the 35-day CI, the reduced lignin alfalfa resulted in lower (P<0.001) yield than controls. Reduced lignin alfalfa lines had 24% and 22% lower (P<0.001) ADL in cuts 1 and 3 than CL lines respectively. . The IVDMD NIR and NDF NIR were greater (p<0.001) in RL than CL lines harvested at the 35-day CI. In alfalfa harvested at the 35-day CI, extent of in vitro gas production and ME content were greater in RL than in CL alfalfa. Although lignin down regulation did reduce ADL, its effect on digestibility was partly counteracted by a reduction of NDF degradability in RL relative to CL alfalfa. Reduced lignin lines had 3.8 % whereas CL had 3.4% (p<0.01) indigestible NDF per unit ADL. The positive effect of lignin down-regulation was more pronounced when intervals between harvests were longer (35-day compared with the 28-day CI). This offers an opportunity to extend harvesting time (CI) for higher yield without compromising the

  19. Engineering of the glycerol decomposition pathway and cofactor regulation in an industrial yeast improves ethanol production.

    Science.gov (United States)

    Zhang, Liang; Tang, Yan; Guo, Zhongpeng; Shi, Guiyang

    2013-10-01

    Glycerol is a major by-product of industrial ethanol production and its formation consumes up to 4 % of the sugar substrate. This study modified the glycerol decomposition pathway of an industrial strain of Saccharomyces cerevisiae to optimize the consumption of substrate and yield of ethanol. This study is the first to couple glycerol degradation with ethanol formation, to the best of our knowledge. The recombinant strain overexpressing GCY1 and DAK1, encoding glycerol dehydrogenase and dihydroxyacetone kinase, respectively, in glycerol degradation pathway, exhibited a moderate increase in ethanol yield (2.9 %) and decrease in glycerol yield (24.9 %) compared to the wild type with the initial glucose concentration of 15 % under anaerobic conditions. However, when the mhpF gene, encoding acetylating NAD⁺-dependent acetaldehyde dehydrogenase from Escherichia coli, was co-expressed in the aforementioned recombinant strain, a further increase in ethanol yield by 5.5 % and decrease in glycerol yield by 48 % were observed for the resultant recombinant strain GDMS1 when acetic acid was added into the medium prior to inoculation compared to the wild type. The process outlined in this study which enhances glycerol consumption and cofactor regulation in an industrial yeast is a promising metabolic engineering strategy to increase ethanol production by reducing the formation of glycerol.

  20. Xplore mRNA assays for the quantification of IL-1 beta and TNF-alpha mRNA in lipopolysaccharide-induced mouse macrophages.

    Science.gov (United States)

    Van Arsdell, S W; Murphy, K P; Pazmany, C; Erickson, D; Burns, C; Moody, M D

    2000-06-01

    Because the accurate measurement of a number of cytokine mRNA transcripts provides valuable knowledge about cytokine gene regulation, we have developed the Xplore assay for the quantification of cytokine mRNA. This microplate-based assay is rapid (under four hours), quantitative over three orders of magnitude and carries no risk of false-positive values from contamination with amplified target. Here, we describe the use of Xplore assays to measure the steady-state mRNA levels of TNF-alpha and IL-1 beta produced by mouse WEHI and J774 macrophage-like cell lines.

  1. Role of exercise-induced brain-derived neurotrophic factor production in the regulation of energy homeostasis in mammals

    DEFF Research Database (Denmark)

    Pedersen, Bente K; Pedersen, Maria; Krabbe, Karen S

    2009-01-01

    identifies BDNF as a player not only in central metabolism, but also in regulating energy metabolism in peripheral organs. Low levels of BDNF are found in patients with neurodegenerative diseases, including Alzheimer's disease and major depression. In addition, BDNF levels are low in obesity...... and independently so in patients with type 2 diabetes. Brain-derived neurotrophic factor is expressed in non-neurogenic tissues, including skeletal muscle, and exercise increases BDNF levels not only in the brain and in plasma, but in skeletal muscle as well. Brain-derived neurotrophic factor mRNA and protein...... expression was increased in muscle cells that were electrically stimulated, and BDNF increased phosphorylation of AMP-activated protein kinase (AMPK) and acetyl coenzyme A carboxylase-beta (ACCbeta) and enhanced fatty oxidation both in vitro and ex vivo. These data identify BDNF as a contraction...

  2. Regulation – Do or Die: An Analysis of Factors Critical to New Product Development in a Regulatory Context

    Directory of Open Access Journals (Sweden)

    Clare O'Dwyer

    2017-04-01

    Full Text Available This study explores new product development in a strict regulatory and historically secretive environment. Adopting a systems perspective and a mixed methods approach in our research, we examine medical device development in Ireland. Findings indicate that the possession of a regulatory strategy expedites the rate of commercialization, so too does the generation of clear product definitions and marketing claims in the earliest developmental phases. Moreover, results suggest that if the regulated industry strengthens its culture for regulation by prioritizing regulation over speed to market, by encouraging cross-functional team collaborations, and by taking a more proactive approach in post-marketing surveillance activities, it has the potential to improve customer satisfaction and enhance product innovation. This study provides unique empirical data enriched by the homogeneity of its sample. It also contributes guidance to practitioners of new product development within a regulatory context.

  3. Characterization, genetic regulation and production of cyanobacterial exopolysaccharides and its applicability for heavy metal removal.

    Science.gov (United States)

    Bhunia, Biswanath; Prasad Uday, Uma Shankar; Oinam, Gunapati; Mondal, Abhijit; Bandyopadhyay, Tarun Kanti; Tiwari, Onkar Nath

    2018-01-01

    Cyanobacteria are uniquely suited for the development of sustainable bioproduction platforms but are currently underutilized due to lack of genetic tools. Exopolysaccharide (EPS) is of significant biotechnological importance due to their technological application in various industries. It has been found that most of the research works are focused on isolation and characterization of new exopolysaccharides from microbial sources. The exopolysaccharides from cyanobacteria have been poorly explored despite their original structural features associated with specific biological and physicochemical properties. However, it could increase in a near future through the use of inexpensive cyanobacterial platform as well as available information on the structural data and specific properties of these biopolymers. This review covers genetic regulation for production of exopolysaccharide, analytical strategies for their characterization, evaluation of structure property relationship and design of extraction protocol from cyanobacterial biomass. In addition applications of exopolysaccharide for removal of heavy metal from wastewater are critically reviewed. Copyright © 2017. Published by Elsevier Ltd.

  4. Regulations governing veterinary medicinal products containing genetically modified organisms in the European community.

    Science.gov (United States)

    Moulin, G

    2005-04-01

    This paper describes particular aspects of the marketing of veterinary medicinal products (VMPs) that contain or consist of genetically modified micro-organisms (GMMs) or genetically modified organisms (GMOs). The regulatory requirements and the procedures applied in the European Union for each phase (pre-marketing, authorisation process, and post-authorisation labelling and monitoring) are explained. In most cases VMPs are subject to both pharmaceutical and GMO regulations. In the early stages of the process, before applications for marketing authorisation are submitted, the assessment of clinical trials and experiments in contained areas is principally the responsibility of national authorities. However, the marketing of all VMPs containing or consisting of GMOs must be authorised at European level, although the national authorities are informed and involved in the assessment process.

  5. Dynamic regulation of fatty acid pools for improved production of fatty alcohols in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Teixeira, Paulo Goncalves; Ferreira, Raphael; Zhou, Yongjin J.

    2017-01-01

    Background: In vivo production of fatty acid-derived chemicals in Saccharomyces cerevisiae requires strategies to increase the intracellular supply of either acyl-CoA or free fatty acids (FFAs), since their cytosolic concentrations are quite low in a natural state for this organism. Deletion...... of the fatty acyl-CoA synthetase genes FAA1 and FAA4 is an effective and straightforward way to disable re-activation of fatty acids and drastically increase FFA levels. However, this strategy causes FFA over-accumulation and consequential release to the extracellular medium, which results in a significant...... decreased by 63% compared with the control strain. Conclusions: Fine-tuning and dynamic regulation of key metabolic steps can be used to improve cell factories when the rates of downstream reactions are limiting. This avoids loss of precursors to the extracellular medium or to competing reactions, hereby...

  6. On a stochastic gene expression with pre-mRNA, mRNA and protein contribution.

    Science.gov (United States)

    Rudnicki, Ryszard; Tomski, Andrzej

    2015-12-21

    In this paper we develop a model of stochastic gene expression, which is an extension of the model investigated in the paper [T. Lipniacki, P. Paszek, A. Marciniak-Czochra, A.R. Brasier, M. Kimmel, Transcriptional stochasticity in gene expression, J. Theor. Biol. 238 (2006) 348-367]. In our model, stochastic effects still originate from random fluctuations in gene activity status, but we precede mRNA production by the formation of pre-mRNA, which enriches classical transcription phase. We obtain a stochastically regulated system of ordinary differential equations (ODEs) describing evolution of pre-mRNA, mRNA and protein levels. We perform mathematical analysis of a long-time behavior of this stochastic process, identified as a piece-wise deterministic Markov process (PDMP). We check exact results using numerical simulations for the distributions of all three types of particles. Moreover, we investigate the deterministic (adiabatic) limit state of the process, when depending on parameters it can exhibit two specific types of behavior: bistability and the existence of the limit cycle. The latter one is not present when only two kinds of gene expression products are considered. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Two Mutations in the Caprine MTHFR 3'UTR Regulated by MicroRNAs Are Associated with Milk Production Traits

    Science.gov (United States)

    Song, Yuxuan; Gao, Teyang; Lei, Yingnan; Cao, Binyun

    2015-01-01

    Background 5,10-Methylenetetrahydrofolate reductase (MTHFR) plays a central role in folate metabolism by irreversibly converting 5,10-methylenetetrahydrofolate to 5-methylenetetrahydrofolate, a predominant circulating form of folate. Folate is reportedly important for milk protein synthesis, and MTHFR may be a key regulatory point of folate metabolism for milk protein synthesis in mammary epithelial cells. Prior to this study, polymorphisms of the MTHFR gene were not associated with milk production traits from a breeding perspective. Single nucleotide polymorphisms (SNPs) at microRNA (miRNA) binding sites (miR-SNPs) can affect gene expression. This study aimed to identify the effects of miR-SNPs (g.2244A>G and g.2264A>G) in the caprine MTHFR 3' UTR on the milk production traits of dairy goats. Results Guanzhong dairy (GD, n = 325) goats were used to detect SNPs in the caprine MTHFR 3' UTR by DNA sequencing. Two novel SNPs (g.2244A>G and g.2264A>G) were identified in the said region. The homozygous haplotype A-G of the SNPs g.2244A>G and g.2264A>G was significantly associated with milk yield and milk protein levels in GD goats (P MTHFR 2244 A → G substitution could increase the binding activity of hsa-miR-1266 with the MTHFR 3' UTR. The MTHFR 2264 A → G substitution could decrease the binding activity of hsa-miR-616 with the MTHFR 3' UTR. In addition, we observed a significant increase in the MTHFR mRNA levels of homozygous haplotype A-G carriers relative to those of homozygous haplotype G-A carriers. These results indicated that both SNPs altered the MTHFR mRNA levels. These altered levels of MTHFR mRNA may account for the association of SNPs with milk production traits. Conclusions This study is the first to report that the g.2244A>G and g.2264A>G polymorphisms were associated with milk production traits in GD goats. Further investigations should explore the underlying miRNA-mediated mechanisms that are modified by the g.2244A>G and g.2264A>G SNPs. The current

  8. Regulation of Rac1 and Reactive Oxygen Species Production in Response to Infection of Gastrointestinal Epithelia.

    Directory of Open Access Journals (Sweden)

    Gerco den Hartog

    2016-01-01

    Full Text Available Generation of reactive oxygen species (ROS during infection is an immediate host defense leading to microbial killing. APE1 is a multifunctional protein induced by ROS and after induction, protects against ROS-mediated DNA damage. Rac1 and NAPDH oxidase (Nox1 are important contributors of ROS generation following infection and associated with gastrointestinal epithelial injury. The purpose of this study was to determine if APE1 regulates the function of Rac1 and Nox1 during oxidative stress. Gastric or colonic epithelial cells (wild-type or with suppressed APE1 were infected with Helicobacter pylori or Salmonella enterica and assessed for Rac1 and NADPH oxidase-dependent superoxide production. Rac1 and APE1 interactions were measured by co-immunoprecipitation, confocal microscopy and proximity ligation assay (PLA in cell lines or in biopsy specimens. Significantly greater levels of ROS were produced by APE1-deficient human gastric and colonic cell lines and primary gastric epithelial cells compared to control cells after infection with either gastric or enteric pathogens. H. pylori activated Rac1 and Nox1 in all cell types, but activation was higher in APE1 suppressed cells. APE1 overexpression decreased H. pylori-induced ROS generation, Rac1 activation, and Nox1 expression. We determined that the effects of APE1 were mediated through its N-terminal lysine residues interacting with Rac1, leading to inhibition of Nox1 expression and ROS generation. APE1 is a negative regulator of oxidative stress in the gastrointestinal epithelium during bacterial infection by modulating Rac1 and Nox1. Our results implicate APE1 in novel molecular interactions that regulate early stress responses elicited by microbial infections.

  9. Circulating Glucagon 1-61 Regulates Blood Glucose by Increasing Insulin Secretion and Hepatic Glucose Production

    Directory of Open Access Journals (Sweden)

    Nicolai J. Wewer Albrechtsen

    2017-11-01

    Full Text Available Glucagon is secreted from pancreatic α cells, and hypersecretion (hyperglucagonemia contributes to diabetic hyperglycemia. Molecular heterogeneity in hyperglucagonemia is poorly investigated. By screening human plasma using high-resolution-proteomics, we identified several glucagon variants, among which proglucagon 1-61 (PG 1-61 appears to be the most abundant form. PG 1-61 is secreted in subjects with obesity, both before and after gastric bypass surgery, with protein and fat as the main drivers for secretion before surgery, but glucose after. Studies in hepatocytes and in β cells demonstrated that PG 1-61 dose-dependently increases levels of cAMP, through the glucagon receptor, and increases insulin secretion and protein levels of enzymes regulating glycogenolysis and gluconeogenesis. In rats, PG 1-61 increases blood glucose and plasma insulin and decreases plasma levels of amino acids in vivo. We conclude that glucagon variants, such as PG 1-61, may contribute to glucose regulation by stimulating hepatic glucose production and insulin secretion.

  10. State Regulation Of Licensing In The Sphere Of Production Of Peat In Russian Federation

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    Pavel A. Byshkov

    2014-12-01

    Full Text Available In the article features of legal regulation of production and state licensing of peat in the modern legislation and laws of the Russian Federation are analyzed. Particular attention is paid to the understanding of peat as a mineral that is important for the country's energy. The process of securing right of the peat use in the modern Russian law is analyzed. The question of the relationship of legislation on the subsoil and water legislation is researched, as well as issues of the Law of the Russian Federation "On Subsoil" and the Water Code of the Russian Federation are correlated. In addition the term "swamp", as the object of peat occurrence is considered. In the broader concept of the "wetland" exists in the Ramsar Convention of the 1971. The order of occurrence, termination, as well as the content of a license for the peat use is analyzed. The license is considered as a special governmental approval, including the prescribed form of the document with the State Emblem of the Russian Federation, as well as text, graphics and other supplements that are an integral part of the license and the fundamental conditions for the subsoil use. Rights and obligations of peat users in Russia are analyzed. Importance of the legal regulation of peat extraction as a common mineral in the Russian law understanding is highlighted.

  11. A genetic strategy to measure circulating Drosophila insulin reveals genes regulating insulin production and secretion.

    Directory of Open Access Journals (Sweden)

    Sangbin Park

    2014-08-01

    Full Text Available Insulin is a major regulator of metabolism in metazoans, including the fruit fly Drosophila melanogaster. Genome-wide association studies (GWAS suggest a genetic basis for reductions of both insulin sensitivity and insulin secretion, phenotypes commonly observed in humans with type 2 diabetes mellitus (T2DM. To identify molecular functions of genes linked to T2DM risk, we developed a genetic tool to measure insulin-like peptide 2 (Ilp2 levels in Drosophila, a model organism with superb experimental genetics. Our system permitted sensitive quantification of circulating Ilp2, including measures of Ilp2 dynamics during fasting and re-feeding, and demonstration of adaptive Ilp2 secretion in response to insulin receptor haploinsufficiency. Tissue specific dissection of this reduced insulin signaling phenotype revealed a critical role for insulin signaling in specific peripheral tissues. Knockdown of the Drosophila orthologues of human T2DM risk genes, including GLIS3 and BCL11A, revealed roles of these Drosophila genes in Ilp2 production or secretion. Discovery of Drosophila mechanisms and regulators controlling in vivo insulin dynamics should accelerate functional dissection of diabetes genetics.

  12. Piperine regulates UCP1 through the AMPK pathway by generating intracellular lactate production in muscle cells.

    Science.gov (United States)

    Kim, Nami; Nam, Miso; Kang, Mi Sun; Lee, Jung Ok; Lee, Yong Woo; Hwang, Geum-Sook; Kim, Hyeon Soo

    2017-01-24

    This study characterizes the human metabolic response to piperine, a curcumin extract, and the details of its underlying molecular mechanism. Using (1)H-NMR-based metabolome analysis, we showed the metabolic effect of piperine on skeletal muscle and found that piperine increased the level of intracellular lactate, an important metabolic intermediate that controls expression of several genes involved in mitochondrial activity. Piperine also induced the phosphorylation of AMP-activated protein kinase (AMPK) and its downstream target, acetyl-CoA carboxylase (ACC), while additionally stimulating glucose uptake in an AMPK dependent manner. Piperine also stimulates the p38 mitogen-activated protein kinase (p38 MAPK), an effect that was reversed by pretreatment with compound C, an AMPK inhibitor. Inhibition of p38 MAPK resulted in no piperine-induced glucose uptake. Increased level of lactate resulted in increased expression of mitochondrial uncoupling protein 1 (UCP1), which regulates energy expenditure, thermogenesis, and fat browning. Knock-down of AMPK blocked piperine-induced UCP1 up-regulation, demonstrating the required role of AMPK in this effect. Taken together, these results suggest that piperine leads to benign metabolic effects by activating the AMPK-p38 MAPK signaling pathway and UCP1 expression by activating intracellular lactate production in skeletal muscle.

  13. Regulation of soybean seed germination through ethylene production in response to reactive oxygen species.

    Science.gov (United States)

    Ishibashi, Yushi; Koda, Yuka; Zheng, Shao-Hui; Yuasa, Takashi; Iwaya-Inoue, Mari

    2013-01-01

    Despite their toxicity, reactive oxygen species (ROS) play important roles in plant cell signalling pathways, such as mediating responses to stress or infection and in programmed cell death, at lower levels. Although studies have indicated that hydrogen peroxide (H(2)O(2)) promotes seed germination of several plants such as Arabidopsis, barley, wheat, rice and sunflower, the role of H(2)O(2) in soybean seed germination is not well known. The aim of this study therefore was to investigate the relationships between ROS, plant hormones and soybean seed germination. An examination was made of soybean seed germination, the expression of genes related to ethylene biosynthesis, endogenous ethylene contents, and the number and area of cells in the root tip, using N-acetylcysteine, an antioxidant, to counteract the effect of ROS. H(2)O(2) promoted germination, which N-acetylcysteine suppressed, suggesting that ROS are involved in the regulation of soybean germination. H(2)O(2) was produced in the embryonic axis after imbibition. N-Acetylcysteine suppressed the expression of genes related to ethylene biosynthesis and the production of endogenous ethylene. Interestingly, ethephon, which is converted to ethylene, and H(2)O(2) reversed the suppression of seed germination by N-acetylcysteine. Furthermore, morphological analysis revealed that N-acetylcysteine suppressed cell elongation at the root tip, and this suppression was also reversed by ethephon or H(2)O(2) treatments, as was the case in germination. In soybean seeds, ROS produced in the embryonic axis after imbibition induce the production of endogenous ethylene, which promotes cell elongation in the root tip. This appears to be how ROS regulate soybean seed germination.

  14. Regulated necrosis-related molecule mRNA expression in humans and mice and in murine acute tissue injury and systemic autoimmunity leading to progressive organ damage, and progressive fibrosis.

    Science.gov (United States)

    Honarpisheh, Mohsen; Desai, Jyaysi; Marschner, Julian A; Weidenbusch, Marc; Lech, Maciej; Vielhauer, Volker; Anders, Hans-Joachim; Mulay, Shrikant R

    2016-12-01

    The species-specific, as well as organ-specific expression of regulated necrosis (RN)-related molecules, is not known. We determined the expression levels of tumour necrosis factor receptor-1 (TNFR1), receptor activated protein kinase (RIPK)1, RIPK3, mixed lineage kinase domain-like (MLKL), CASP8, Fas-associated protein with death domain (FADD), cellular inhibitor of apoptosis protein (CIAP)1, CIAP2, glutathione peroxidase-4 (GPX4), cyclophilin D (CYPD), CASP1, NLRP3 and poly(ADP-ribose) polymerase-1 (PARP1) in human and mouse solid organs. We observed significant differences in expression of these molecules between human and mice. In addition, we characterized their expression profiles in acute as well as persistent tissue injury and chronic tissue remodelling using acute and chronic kidney injury models. We observed that the degree and pattern of induction of RN-related molecules were highly dependent on the trigger and disease pathogenesis. Furthermore, we studied their expression patterns in mice with lupus-like systemic autoimmunity, which revealed that the expression of MLKL, GPX4 and PARP1 significantly increased in the spleen along disease progression and CASP1, RIPK1, RIPK3 and CYPD were higher at the earlier stages but were significantly decreased in the later stages. In contrast, in the kidney, the expression of genes involved in pyroptosis, e.g. NLRP3 and CASP1 were significantly increased and TNFR1, RIPK1, RIPK3, CIAP1/2 and GPX4 were significantly decreased along the progression of lupus nephritis (LN). Thus, the organ- and species-specific expression of RN-related molecules should be considered during designing experiments, interpreting the results as well as extrapolating the conclusions from one species or organ to another species or organ respectively. © 2016 The Author(s).

  15. Strain-Level Differences in Porphyrin Production and Regulation in Propionibacterium acnes Elucidate Disease Associations

    Science.gov (United States)

    Johnson, Tremylla; Kang, Dezhi; Barnard, Emma

    2016-01-01

    ABSTRACT Propionibacterium acnes is an important skin commensal, but it is also considered a pathogenic factor in several diseases including acne vulgaris, the most common skin disease. While previous studies have revealed P. acnes strain-level differences in health and disease associations, the underlying molecular mechanisms remain unknown. Recently, we demonstrated that vitamin B12 supplementation increases P. acnes production of porphyrins, a group of proinflammatory metabolites important in acne development (D. Kang, B. Shi, M. C. Erfe, N. Craft, and H. Li, Sci. Transl. Med. 7:293ra103, 2015, doi:10.1126/scitranslmed.aab2009). In this study, we compared the porphyrin production and regulation of multiple P. acnes strains. We revealed that acne-associated type IA-2 strains inherently produced significantly higher levels of porphyrins, which were further enhanced by vitamin B12 supplementation. On the other hand, health-associated type II strains produced low levels of porphyrins and did not respond to vitamin B12. Using a small-molecule substrate and inhibitor, we demonstrated that porphyrin biosynthesis was modulated at the metabolic level. We identified a repressor gene (deoR) of porphyrin biosynthesis that was carried in all health-associated type II strains, but not in acne-associated type IA-2 strains. The expression of deoR suggests additional regulation of porphyrin production at the transcriptional level in health-associated strains. Our findings provide one potential molecular mechanism for the different contributions of P. acnes strains to skin health and disease and support the role of vitamin B12 in acne pathogenesis. Our study emphasizes the importance of understanding the role of the commensal microbial community in health and disease at the strain level and suggests potential utility of health-associated P. acnes strains in acne treatment. IMPORTANCE Propionibacterium acnes is a dominant bacterium residing on skin, and it has been thought

  16. Long non-coding RNA Lethe regulates hyperglycemia-induced reactive oxygen species production in macrophages.

    Directory of Open Access Journals (Sweden)

    Carlos Zgheib

    Full Text Available Type 2 diabetes mellitus is a complex, systemic metabolic disease characterized by insulin resistance and resulting hyperglycemia, which is associated with impaired wound healing. The clinical complications associated with hyperglycemia are attributed, in part, to the increased production of reactive oxygen species (ROS. Recent studies revealed that long non-coding RNAs (lncRNAs play important regulatory roles in many biological processes. Specifically, lncRNA Lethe has been described as exhibiting an anti-inflammatory effect by binding to the p65 subunit of NFκB and blocking its binding to DNA and the subsequent activation of downstream genes. We therefore hypothesize that dysregulation of Lethe's expression plays a role in hyperglycemia-induced ROS production. To test our hypothesis, we treated RAW264.7 macrophages with low glucose (5 mM or high glucose (25 mM for 24h. High glucose conditions significantly induced ROS production and NOX2 gene expression in RAW cells, while significantly decreasing Lethe gene expression. Overexpression of Lethe in RAW cells eliminated the increased ROS production induced by high glucose conditions, while also attenuating the upregulation of NOX2 expression. Similar results was found also in non-diabetic and diabetic primary macrophage, bone marrow derived macrophage (BMM. Furthermore, overexpression of Lethe in RAW cells treated with high glucose significantly reduced the translocation of p65-NFkB to the nucleus, which resulted in decreased NOX2 expression and ROS production. Interestingly, these findings are consistent with the decreased Lethe gene expression and increased NOX2 gene expression observed in a mouse model of diabetic wound healing. These findings provide the first evidence that lncRNA Lethe is involved in the regulation of ROS production in macrophages through modulation of NOX2 gene expression via NFκB signaling. Moreover, this is the first report to describe a role of lncRNAs, in particular

  17. Long non-coding RNA Lethe regulates hyperglycemia-induced reactive oxygen species production in macrophages.

    Science.gov (United States)

    Zgheib, Carlos; Hodges, Maggie M; Hu, Junyi; Liechty, Kenneth W; Xu, Junwang

    2017-01-01

    Type 2 diabetes mellitus is a complex, systemic metabolic disease characterized by insulin resistance and resulting hyperglycemia, which is associated with impaired wound healing. The clinical complications associated with hyperglycemia are attributed, in part, to the increased production of reactive oxygen species (ROS). Recent studies revealed that long non-coding RNAs (lncRNAs) play important regulatory roles in many biological processes. Specifically, lncRNA Lethe has been described as exhibiting an anti-inflammatory effect by binding to the p65 subunit of NFκB and blocking its binding to DNA and the subsequent activation of downstream genes. We therefore hypothesize that dysregulation of Lethe's expression plays a role in hyperglycemia-induced ROS production. To test our hypothesis, we treated RAW264.7 macrophages with low glucose (5 mM) or high glucose (25 mM) for 24h. High glucose conditions significantly induced ROS production and NOX2 gene expression in RAW cells, while significantly decreasing Lethe gene expression. Overexpression of Lethe in RAW cells eliminated the increased ROS production induced by high glucose conditions, while also attenuating the upregulation of NOX2 expression. Similar results was found also in non-diabetic and diabetic primary macrophage, bone marrow derived macrophage (BMM). Furthermore, overexpression of Lethe in RAW cells treated with high glucose significantly reduced the translocation of p65-NFkB to the nucleus, which resulted in decreased NOX2 expression and ROS production. Interestingly, these findings are consistent with the decreased Lethe gene expression and increased NOX2 gene expression observed in a mouse model of diabetic wound healing. These findings provide the first evidence that lncRNA Lethe is involved in the regulation of ROS production in macrophages through modulation of NOX2 gene expression via NFκB signaling. Moreover, this is the first report to describe a role of lncRNAs, in particular Lethe, in

  18. Analysis of mRNA recognition by human thymidylate synthase.

    Science.gov (United States)

    Brunn, Nicholas D; Dibrov, Sergey M; Kao, Melody B; Ghassemian, Majid; Hermann, Thomas

    2014-12-23

    Expression of hTS (human thymidylate synthase), a key enzyme in thymidine biosynthesis, is regulated on the translational level through a feedback mechanism that is rarely found in eukaryotes. At low substrate concentrations, the ligand-free enzyme binds to its own mRNA and stabilizes a hairpin structure that sequesters the start codon. When in complex with dUMP (2'-deoxyuridine-5'-monophosphate) and a THF (tetrahydrofolate) cofactor, the enzyme adopts a conformation that is unable to bind and repress expression of mRNA. Here, we have used a combination of X-ray crystallography, RNA mutagenesis and site-specific cross-linking studies to investigate the molecular recognition of TS mRNA by the hTS enzyme. The interacting mRNA region was narrowed to the start codon and immediately flanking sequences. In the hTS enzyme, a helix-loop-helix domain on the protein surface was identified as the putative RNA-binding site.

  19. Low-level lasers on microRNA and uncoupling protein 2 mRNA levels in human breast cancer cells

    Science.gov (United States)

    Canuto, K. S.; Teixeira, A. F.; Rodrigues, J. A.; Paoli, F.; Nogueira, E. M.; Mencalha, A. L.; Fonseca, A. S.

    2017-06-01

    MicroRNA is short non-coding RNA and is a mediator of post-transcriptional regulation of gene expression. In addition, uncoupling proteins (UCPs) regulate thermogenesis, metabolic and energy balance, and decrease reactive oxygen species production. Both microRNA and UCP2 expression can be altered in cancer cells. At low power, laser wavelength, frequency, fluence and emission mode deternube photobiological responses, which are the basis of low-level laser therapy. There are few studies on miRNA and UCP mRNA levels after low-level laser exposure on cancer cells. In this work, we evaluate the micrRNA (mir-106b and mir-15a) and UCP2 mRNA levels in human breast cancer cells exposed to low-level lasers. MDA-MB-231 human breast cancer cells were exposed to low-level red and infrared lasers, total RNA was extracted for cDNA synthesis and mRNA levels by real time quantitative polymerase chain reaction were evaluated. Data show that mir-106b and mir-15a relative levels are not altered, but UCP2 mRNA relative levels are increased in MDA-MB-231 human breast cancer cells exposed to low-level red and infrared lasers at fluences used in therapeutic protocols.

  20. Translational repression of mRNA for eucaryotic elongation factors in Friend erythroleukemia cells.

    Science.gov (United States)

    Slobin, L I; Jordan, P

    1984-11-15

    Poly(A)-containing mRNA was prepared from polyribosomes and postpolyribosomal messenger ribonucleoprotein particles (mRNP) from Friend erythroleukemic cells. Both mRNA types were translated in vitro and the 35S-labeled translation products examined by two-dimensional gel electrophoresis. Among the most abundant untranslated mRNA species was the mRNA coding for eucaryotic elongation factor Tu (eEF-Tu). In addition, the mRNA for eucaryotic elongation factor Ts was also present in Friend cells in untranslated form. Calculations based on translation assays indicate that eEF-Tu represents about 15% of the translation products of RNP mRNA and that approximately 40% of the eEF-Tu synthesized in vitro is encoded by translationally repressed mRNA. This repressed mRNA can be activated by addition of cycloheximide to cell cultures. At the level of 0.1 micrograms/ml, cycloheximide was found to inhibit cellular protein synthesis by about 50% while augmenting the relative rate of eEF-Tu synthesis 1.6-fold. This result suggested that eEF-Tu mRNA might initiate poorly. However, addition of supersaturating levels of mRNA to a reticulocyte lysate augmented eEF-Tu synthesis about twofold, while generally depressing the synthesis of other proteins by about 40%. Thus the storage of large amounts of eEF-Tu mRNA in vivo is unlikely to be due directly to the ineffectiveness of the mRNA in competing for the initiation machinery of the cell. The results presented in this report suggest that the supply of active eEF-Tu in erythroleukemic cells is controlled, at least in part, by a translational mechanism.

  1. SlyA regulates phytotoxin production and virulence in Dickeya zeae EC1.

    Science.gov (United States)

    Zhou, Jia-Nuan; Zhang, Hai-Bao; Lv, Ming-Fa; Chen, Yu-Fan; Liao, Li-Sheng; Cheng, Ying-Ying; Liu, Shi-Yin; Chen, Shao-Hua; He, Fei; Cui, Zi-Ning; Jiang, Zi-De; Chang, Chang-Qing; Zhang, Lian-Hui

    2016-12-01

    Dickeya zeae is a causal agent of rice root rot disease. The pathogen is known to produce a range of virulence factors, including phytotoxic zeamines and extracellular enzymes, but the mechanisms of virulence regulation remain vague. In this study, we identified a SlyA/MarR family transcription factor SlyA in D. zeae strain EC1. Disruption of slyA significantly decreased zeamine production, enhanced swimming and swarming motility, reduced biofilm formation and significantly decreased pathogenicity on rice. Quantitative polymerase chain reaction (qPCR) analysis confirmed the role of SlyA in transcriptional modulation of a range of genes associated with bacterial virulence. In trans expression of slyA in expI mutants recovered the phenotypes of motility and biofilm formation, suggesting that SlyA is downstream of the acylhomoserine lactone-mediated quorum sensing pathway. Taken together, the findings from this study unveil a key transcriptional regulatory factor involved in the modulation of virulence factor production and overall pathogenicity of D. zeae EC1. © 2016 BSPP and John Wiley & Sons Ltd.

  2. Toxicant exposure from smoking a little cigar: further support for product regulation.

    Science.gov (United States)

    Pickworth, Wallace B; Rosenberry, Zachary R; Koszowski, Bartosz

    2017-05-01

    Although numerous studies have documented the prevalence and increasing use of little cigars and other cigar products, the present study is the first direct, head-to-head laboratory comparison of little cigar and cigarette smoking. The study addressed a fundamental objective to compare exposure and use characteristics of little cigar and cigarette smoking. Smoking patterns, toxicant exposure and subjective measures were collected and analysed in 21 adults after smoking a little cigar (Winchester) and a cigarette (own brand). Participants were dual users of little cigars and cigarettes. Similar to cigarettes, little cigars delivered substantial nicotine and relatively more carbon monoxide. Puff volume, puff duration and time to smoke were significantly greater after cigarettes, but the temporal pattern of smoking more intensively at the beginning was similar in little cigars and cigarettes. Both little cigars and cigarettes reduced urge to smoke. Participants consistently mentioned that the lower cost of little cigars was a reason for initiation and continuation of their use. The results support the notion that regulation of little cigars is appropriate in light of public health considerations. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  3. The regulation of exopolysaccharide production is important at two levels of nodule development in Rhizobium meliloti.

    Science.gov (United States)

    Ozga, D A; Lara, J C; Leig, J A

    1994-01-01

    We show that two exopolysaccharide overproducing Tn5 mutants of Rhizobium meliloti, exoR and exoS, have distinct symbiotic defects. While the exoR mutant is unable to colonize nodules, the exoS mutant retains that ability but varies in its ability to produce nitrogen-fixing nodules. We correlate these defects with different degrees of exopolysaccharide overproduction and growth impairment. We further show that the exoR mutant is able to enter developing infection threads but is unable to invade nodule cells. The exoR mutant gives rise to spontaneous pseudorevertants containing second-site suppressor mutations that decrease exopolysaccharide synthesis. These pseudorevertants form nitrogen-fixing nodules. Although the suppressor mutations have the opposite effect on exopolysaccharide production compared to the exoS::Tn5 mutation, they consistently map to the exoS::Tn5 region and belong to the same genetic complementation group as defined by transposon insertion mutations. The effect of the suppressor mutations on exopolysaccharide production is correlated with effects on the expression of exo genes involved in exopolysaccharide synthesis. Finally, we provide evidence that the exoR gene is not required for the regulation of exopolysaccharide synthesis by ammonia.

  4. Orthologs of the archaeal isopentenyl phosphate kinase regulate terpenoid production in plants.

    Science.gov (United States)

    Henry, Laura K; Gutensohn, Michael; Thomas, Suzanne T; Noel, Joseph P; Dudareva, Natalia

    2015-08-11

    Terpenoids, compounds found in all domains of life, represent the largest class of natural products with essential roles in their hosts. All terpenoids originate from the five-carbon building blocks, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), which can be derived from the mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways. The absence of two components of the MVA pathway from archaeal genomes led to the discovery of an alternative MVA pathway with isopentenyl phosphate kinase (IPK) catalyzing the final step, the formation of IPP. Despite the fact that plants contain the complete classical MVA pathway, IPK homologs were identified in every sequenced green plant genome. Here, we show that IPK is indeed a member of the plant terpenoid metabolic network. It is localized in the cytosol and is coexpressed with MVA pathway and downstream terpenoid network genes. In planta, IPK acts in parallel with the MVA pathway and plays an important role in regulating the formation of both MVA and MEP pathway-derived terpenoid compounds by controlling the ratio of IP/DMAP to IPP/DMAPP. IP and DMAP can also competitively inhibit farnesyl diphosphate synthase. Moreover, we discovered a metabolically available carbon source for terpenoid formation in plants that is accessible via IPK overexpression. This metabolite reactivation approach offers new strategies for metabolic engineering of terpenoid production.

  5. Chrysosplenetin inhibits artemisinin efflux in P-gp-over-expressing Caco-2 cells and reverses P-gp/MDR1 mRNA up-regulated expression induced by artemisinin in mouse small intestine.

    Science.gov (United States)

    Ma, Liping; Wei, Shijie; Yang, Bei; Ma, Wei; Wu, Xiuli; Ji, Hongyan; Sui, Hong; Chen, Jing

    2017-12-01

    CYP3A4 and P-gp together form a highly efficient barrier for orally absorbed drugs and always share the same substrates. Our previous work revealed that chrysosplenetin (CHR) significantly augmented the rat plasma level and anti-malarial efficacy of artemisinin (ART), partially due to the uncompetitive inhibition effect of CHR on rat CYP3A. But the impact of CHR on P-gp is still unknown. The present study investigates whether CHR interferes with P-gp-mediated efflux of ART and elucidates the underlying mechanism. P-gp-over-expressing Caco-2 cells were treated with ART (10 μM) or ART-CHR (1:2, 10:20 μM) in ART bidirectional transport experiment. ART concentration was determined by UHPLC-MS/MS method. Healthy male ICR mice were randomly divided into nine groups (n = 6) including negative control (0.5% CMC-Na solution, 13 mL/kg), ART alone (40 mg/kg), verapamil (positive control, 40 mg/kg), ART-verapamil (1:1, 40:40 mg/kg), CHR alone (80 mg/kg), ART-CHR (1:0.1, 40:4 mg/kg), ART-CHR (1:1, 40:40 mg/kg), ART-CHR (1:2, 40:80 mg/kg) and ART-CHR (1:4, 40:160 mg/kg). The drugs were administrated intragastrically for seven consecutive days. MDR1 and P-gp expression levels in mice small intestine were examined by performing RT-PCR and western blot analysis. ABC coupling ATPase activity was also determined. After combined with CHR (1:2), Papp (AP-BL) and Papp (BL-AP) of ART changed to 4.29 × 10 (-) (8) (increased 1.79-fold) and 2.85 × 10 (-) (8 )cm/s (decreased 1.24-fold) from 2.40 × 10 (-) (8) and 3.54 × 10 (-) (8 )cm/s, respectively. Efflux ratio (PBA/PAB) declined 2.21-fold (p P-gp levels compared with vehicle, while CHR in combination ratio of 0:1, 0.1:1, 1:1, 2:1 and 4:1 with ART, reversed them to normal levels as well as negative control (p p P-gp activity and reverse the up-regulated P-gp and MDR1 levels induced by ART. It suggested that CHR potentially can be used as a P-gp reversal agent to

  6. Regulation of IgA production by naturally occurring TNF/iNOS-producing dendritic cells.

    Science.gov (United States)

    Tezuka, Hiroyuki; Abe, Yukiko; Iwata, Makoto; Takeuchi, Hajime; Ishikawa, Hiromichi; Matsushita, Masayuki; Shiohara, Tetsuo; Akira, Shizuo; Ohteki, Toshiaki

    2007-08-23

    Immunoglobulin-A has an irreplaceable role in the mucosal defence against infectious microbes. In human and mouse, IgA-producing plasma cells comprise approximately 20% of total plasma cells of peripheral lymphoid tissues, whereas more than 80% of plasma cells produce IgA in mucosa-associated lymphoid tissues (MALT). One of the most biologically important and long-standing questions in immunology is why this 'biased' IgA synthesis takes place in the MALT but not other lymphoid organs. Here we show that IgA class-switch recombination (CSR) is impaired in inducible-nitric-oxide-synthase-deficient (iNOS-/-; gene also called Nos2) mice. iNOS regulates the T-cell-dependent IgA CSR through expression of transforming growth factor-beta receptor, and the T-cell-independent IgA CSR through production of a proliferation-inducing ligand (APRIL, also called Tnfsf13) and a B-cell-activating factor of the tumour necrosis factor (TNF) family (BAFF, also called Tnfsf13b). Notably, iNOS is preferentially expressed in MALT dendritic cells in response to the recognition of commensal bacteria by toll-like receptor. Furthermore, adoptive transfer of iNOS+ dendritic cells rescues IgA production in iNOS-/- mice. Further analysis revealed that the MALT dendritic cells are a TNF-alpha/iNOS-producing dendritic-cell subset, originally identified in mice infected with Listeria monocytogenes. The presence of a naturally occurring TNF-alpha/iNOS-producing dendritic-cell subset may explain the predominance of IgA production in the MALT, critical for gut homeostasis.

  7. Prostaglandin D2 production in FM55 melanoma cells is regulated by α-melanocyte-stimulating hormone and is not related to melanin production

    Science.gov (United States)

    Masoodi, Mojgan; Nicolaou, Anna; Gledhill, Karl; Rhodes, Lesley E; Tobin, Desmond J; Thody, Anthony J

    2010-01-01

    This study shows that prostaglandins in human FM55 melanoma cells and epidermal melanocytes are produced by COX-1. Prostaglandin production in FM55 melanoma cells was unrelated to that of melanin suggesting that the two processes can occur independently. α-Melanocyte-stimulating hormone, which had no effect on melanin production in FM55 cells, stimulated PGD2 production in these cells without affecting PGE2. While cAMP pathways may be involved in regulating PGD2 production, our results suggest that α-MSH acts independently of cAMP, possibly by regulating the activity of lipocalin-type PGD synthase. This α-MSH-mediated effect may be associated with its role as an immune modulator. PMID:20482620

  8. Endothelial surface glycocalyx can regulate flow-induced nitric oxide production in microvessels in vivo.

    Directory of Open Access Journals (Sweden)

    Wanyi Yen

    Full Text Available Due to its unique location, the endothelial surface glycocalyx (ESG at the luminal side of the microvessel wall may serve as a mechano-sensor and transducer of blood flow and thus regulate endothelial functions. To examine this role of the ESG, we used fluorescence microscopy to measure nitric oxide (NO production in post-capillary venules and arterioles of rat mesentery under reduced (low and normal (high flow conditions, with and without enzyme pretreatment to remove heparan sulfate (HS of the ESG and in the presence of an endothelial nitric oxide synthase (eNOS inhibitor, NG-monomethyl-L-arginine (L-NMMA. Rats (SD, 250-300 g were anesthetized. The mesentery was gently taken out from the abdominal cavity and arranged on the surface of a glass coverslip for the measurement. An individual post-capillary venule or arteriole was cannulated and loaded for 45 min with 5 μM 4, 5-Diaminofluorescein diacetate, a membrane permeable fluorescent indictor for NO, then the NO production was measured for ~10 min under a low flow (~300 μm/s and for ~60 min under a high flow (~1000 μm/s. In the 15 min after switching to the high flow, DAF-2-NO fluorescence intensity increased to 1.27-fold of its baseline, DAF-2-NO continuously increased under the high flow, to 1.53-fold of its baseline in 60 min. Inhibition of eNOS by 1 mM L-NMMA attenuated the flow-induced NO production to 1.13-fold in 15 min and 1.30-fold of its baseline in 60 min, respectively. In contrast, no significant increase in NO production was observed after switching to the high flow for 60 min when 1 h pretreatment with 50 mU/mL heparanase III to degrade the ESG was applied. Similar NO production was observed in arterioles under low and high flows and under eNOS inhibition. Our results suggest that ESG participates in endothelial cell mechanosensing and transduction through its heparan sulfate to activate eNOS.

  9. Sodium houttuyfonate affects production of N-acyl homoserine lactone and quorum sensing-regulated genes expression in Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Daqiang eWu

    2014-11-01

    Full Text Available Quorum sensing (QS is a means of cell-to-cell communication that uses diffusible signaling molecules that are sensed by the population to determine population density, thus allowing co-ordinate gene regulation in response to population density. In Pseudomonas aeruginosa, production of the QS signaling molecule, N-acyl homoserine lactone (AHL, co-ordinates expression of key factors of pathogenesis, including biofilm formation and toxin secretion. It is predicted that the inhibition of AHL sensing would provide an effective clinical treatment to reduce the expression of virulence factors and increase the effectiveness of antimicrobial agents. We previously demonstrated that sodium houttuyfonate (SH, commonly used in traditional Chinese medicine to treat infectious diseases, can effectively inhibit QS-regulated processes, including biofilm formation. Here, using a model system, we demonstrate that SH causes the dose-dependent inhibition of AHL production, through down-regulation of the AHL biosynthesis gene, lasI. Addition of SH also resulted in down-regulation of expression of the AHL sensor and transcriptional regulator, LasR, and inhibited the production of the QS-regulated virulence factors, pyocyanin and LasA. These results suggest that the antimicrobial activity of SH may be due to its ability to disrupt QS in P. aeruginosa.

  10. Two Adjacent and Similar TetR Family Transcriptional Regulator Genes, SAV577 and SAV576, Co-Regulate Avermectin Production in Streptomyces avermitilis

    OpenAIRE

    Jia Guo; Xuan Zhang; Zhi Chen; Ying Wen; Jilun Li

    2014-01-01

    Streptomyces avermitilis is an important bacterial species used for industrial production of avermectins, a family of broad-spectrum anthelmintic agents. We previously identified the protein SAV576, a TetR family transcriptional regulator (TFR), as a downregulator of avermectin biosynthesis that acts by controlling transcription of its major target gene SAV575 (which encodes cytochrome P450/NADPH-ferrihemoprotein reductase) and ave genes. SAV577, another TFR gene, encodes a SAV577 protein tha...

  11. Becoming a refinery leader by changing operations to match new product quality regulations

    Energy Technology Data Exchange (ETDEWEB)

    Mendez, Maryro P. [KBC Advanced Technologies, Surrey (United Kingdom)

    2012-07-01

    Global environmental concerns have forced the automotive and oil industries to increase efficiency and reduce emissions, which has led to the tightening of fuels specifications around the world. The implementation of ultra-low sulphur transport fuels has become a worldwide trend with growing momentum. Compliance with these standards requires the refiner to make decisions in advance of the implementation date. Therefore, in order to make changes on time, refiners are currently assessing options and changes required to comply with regulations by 2016. Similar regulations have been implemented in Europe already and KBC has the methodology and experience to assess the existing refinery configuration, unit capability and facility infrastructure to provide the basis for decision making. This paper focus on KBC's methodology which looks at the molecular management needed to produce the low levels of sulfur and toxic required in today's refined products. A key element to the study is the use of Petro-SIM for development of a representative detailed non-linear model, of the refinery which has been used to test ideas and configurations and help confirm the Refinery LP development activities. Using a Petro-SIM representation, KBC is able to make an assessment of the impact of imposing Tier 3 gasoline (sulphur to < 10 ppm) on the refinery, examining critical blending constraints, unused stream qualities and quantities and likely type and scale of capital investment that would be required while optimising unit operations and maximising margins. This paper illustrates KBC's current thinking based on studies done to date to review clean fuels and Tier 3 specifications options for specific refinery configurations. (author)

  12. Lessons from the 'Humanitarian Golden Rice' project: regulation prevents development of public good genetically engineered crop products.

    Science.gov (United States)

    Potrykus, Ingo

    2010-11-30

    Compared to a non-Genetically Engineered (GE) variety, the deployment of Golden Rice has suffered from a delay of at least ten years. The cause of this delay is exclusively GE-regulation. Considering the potential impact of Golden Rice on the reduction in vitamin A-malnutrition, this delay is responsible for an unjustifiable loss of millions of lives, mostly children and women. GE-regulation is also responsible for the fact that no public institution can deliver a public good GE-product and that thus we have a de facto monopoly in favour of a few potent industries. Considering the forgone benefits from prevented public good GE-products, GE-regulation is responsible for hundreds of millions of lives, all of them, of course, in developing countries. As there is no scientific justification for present GE-regulation, and as it has, so far, not prevented any harm, our society has the urgent responsibility to reconsider present regulation, which is based on an extreme interpretation of the precautionary principle, and change it to science-based regulation on the basis of traits instead of technology. GE-technology has an unprecedented safety record and is far more precise and predictable than any other 'traditional' and unregulated breeding technology. Not to change GE-regulation to a scientific basis is considered by the author 'a crime against humanity'. Copyright © 2010 Elsevier B.V. All rights reserved.

  13. Interleukin-21 mRNA expression during virus infections

    DEFF Research Database (Denmark)

    Holm, Christian; Nyvold, Charlotte Guldborg; Paludan, Søren Riis

    2006-01-01

    and activational effects of IL-21 on different leukocytes come into play in vivo in an immune response has so far not been fully investigated. We show here for the first time in vivo, that IL-21 mRNA is produced in the spleen when mice are challenged with herpes simplex virus type 2 (HSV-2) or lymphocytic...... choriomeningitis virus (LCMV). We show in HSV-2 challenged mice that this production takes place in CD4+ T cell fractions and is absent in CD4+ T cell-depleted fractions. We also show that the peak of IL-21 mRNA production in both the HSV-2 and LCMV-challenged mice coincides with the onset of the adaptive immune...... response. Thus, our data suggest a role for IL-21 in the early stages of adaptive immune response against virus infections....

  14. Translatability of rat kidney mRNA after mercury administration.

    Science.gov (United States)

    Samji, S; Kuliszewski, M J; Girgis, G R; Nicholls, D M

    1985-09-01

    Young male rats received an intraperitoneal injection of 0.5 mg HgCl2/kg body weight and 16 h later the kidneys were removed and homogenized to prepare the polysomal fraction from which the poly(A)+ RNA was obtained. The activity of this fraction was assessed by translating the poly(A)+ RNA in a mRNA-dependent rabbit reticulocyte lysate and the activity was markedly elevated relative to preparations from control rat kidneys. The incorporation of labelled leucine and cysteine, but not phenylalanine, into a low molecular weight protein (approximately 10 000 as judged by denaturing polyacrylamide gel electrophoresis) accounted for the increased mRNA activity. The mobility during electrophoresis of the denatured labelled product and carboxymethylated product, as well as their acidic isoelectric points, provided evidence that it is metallothionein mRNA which exhibits increased translatability in preparations derived from mercury-treated rats.

  15. Complex marine natural products as potential epigenetic and production regulators of antibiotics from a marine Pseudomonas aeruginosa

    Science.gov (United States)

    Marine microbes are capable of producing secondary metabolites for defense and competition. Factors exerting an impact on secondary metabolite production of microbial communities included bioactive natural products and co-culturing. These external influences may have practical applications such as ...

  16. IL-1β directly suppress ghrelin mRNA expression in ghrelin-producing cells.

    Science.gov (United States)

    Bando, Mika; Iwakura, Hiroshi; Ueda, Yoko; Ariyasu, Hiroyuki; Inaba, Hidefumi; Furukawa, Yasushi; Furuta, Hiroto; Nishi, Masahiro; Akamizu, Takashi

    2017-05-15

    In animal models, ghrelin production is suppressed by LPS administration. To elucidate the detailed molecular mechanisms involved in the phenomenon, we investigated the effects of LPS and LPS-inducible cytokines, including TNF-α, IL-1β, and IL-6, on the expression of ghrelin in the ghrelin-producing cell line MGN3-1. These cells expressed IL-1R, and IL-1β significantly suppressed ghrelin mRNA levels. The suppressive effects of IL-1β were attenuated by knockdown of IKKβ, suggesting the involvement of the NF-κB pathway. These results suggested that IL-1β is a major regulator of ghrelin expression during inflammatory processes. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Two adjacent and similar TetR family transcriptional regulator genes, SAV577 and SAV576, co-regulate avermectin production in Streptomyces avermitilis.

    Directory of Open Access Journals (Sweden)

    Jia Guo

    Full Text Available Streptomyces avermitilis is an important bacterial species used for industrial production of avermectins, a family of broad-spectrum anthelmintic agents. We previously identified the protein SAV576, a TetR family transcriptional regulator (TFR, as a downregulator of avermectin biosynthesis that acts by controlling transcription of its major target gene SAV575 (which encodes cytochrome P450/NADPH-ferrihemoprotein reductase and ave genes. SAV577, another TFR gene, encodes a SAV577 protein that displays high amino acid homology with SAV576. In this study, we examined the effect of SAV577 on avermectin production and the relationships between SAV576 and SAV577. SAV577 downregulated avermectin biosynthesis indirectly, similarly to SAV576. SAV576 and SAV577 both directly repressed SAV575 transcription, and reciprocally repressed each other's expression. SAV575 transcription levels in various S. avermitilis strains were correlated with avermectin production levels. DNase I footprinting and electrophoretic mobility shift assays indicated that SAV576 and SAV577 compete for the same binding regions, and that DNA-binding affinity of SAV576 is much stronger than that of SAV577. GST pull-down assays revealed no direct interaction between the two proteins. Taken together, these findings suggest that SAV577 regulates avermectin production in S. avermitilis by a mechanism similar to that of SAV576, and that the role of SAV576 is dominant over that of SAV577. This is the first report of two adjacent and similar TFR genes that co-regulate antibiotic production in Streptomyces.

  18. Regulation of EPS production in Lactobacillus casei LC2W through metabolic engineering.

    Science.gov (United States)

    Li, N; Huang, Y; Liu, Z; You, C; Guo, B

    2015-12-01

    Lactobacillus casei LC2W is an exopolysaccharide(EPS)-producing strain with probiotic effects. The low efficiency and unclear regulation mechanism of EPS biosynthesis have become main constraints for its application in food industry. To investigate the major rate-limiting factors of EPS biosynthesis and to improve its yield, metabolic engineering was applied to this strain. Eight relevant genes related to central metabolism, sugar-nucleotides supply, glycosyltransferase and cofactor engineering were cloned and overexpressed. The results suggested that nox, pfk, rfbB and galT genes were the largest contributors to EPS biosynthesis in this study, which elevated EPS yield by 46·0, 20, 17·4 and 19·6% respectively. Notably, under aerobic condition which was not a suitable condition for lactobacilli to grow in, recombinant strain LC-nox achieved the highest EPS yield of 263·7 mg l(-1) , which was increased by 75% compared to that of the starting strain. The oxygen stress was excluded since the phenomenon was not observed in the control strain under the same condition. Therefore, it was probably that higher NADH oxidase activity led to a decreased NADH availability and reduced lactate concentration, which resulted in the elevation of EPS yield. This study contributed to the understanding of EPS biosynthesis in Lact. casei through metabolic engineering and provided a starting point for introducing cofactor engineering into this strain. Overexpression of NADH oxidase was found to have a most significant effect on the EPS production. It is the first report that EPS could be accumulated to such a high level under aerobic condition in lactobacilli. Our results provided a novel strategy for the improvement of EPS production in lactic acid bacteria. © 2015 The Society for Applied Microbiology.

  19. Iron-Regulated Expression of Alginate Production, Mucoid Phenotype, and Biofilm Formation by Pseudomonas aeruginosa

    Science.gov (United States)

    Wiens, Jacinta R.; Vasil, Adriana I.; Schurr, Michael J.; Vasil, Michael L.

    2014-01-01

    ABSTRACT Pseudomonas aeruginosa strains of non-cystic fibrosis (non-CF) origin do not produce significant amounts of extracellular alginate and are nonmucoid. In CF, such isolates can become mucoid through mutation of one of the genes (mucA, mucB, mucC, or mucD) that produce regulatory factors that sequester AlgU, required for increased expression of alginate genes. Mutation of the muc genes in the nonmucoid PAO1, PA14, PAKS-1, and Ps388 strains led to increased levels of extracellular alginate and an obvious mucoid phenotype, but only under iron-limiting growth conditions (≤5 µM), not under iron-replete conditions (≥10 µM). In contrast, >50% of P. aeruginosa isolates from chronic CF pulmonary infections expressed increased levels of alginate and mucoidy both under iron-limiting and iron-replete conditions (i.e., iron-constitutive phenotype). No single iron regulatory factor (e.g., Fur, PvdS) was associated with this loss of iron-regulated alginate expression and mucoidy in these CF isolates. However, the loss of only pyoverdine production, or its uptake, abrogated the ability of P. aeruginosa to produce a robust biofilm that represents the Psl-type of biofilm. In contrast, we show that mutation of the pyoverdine and pyochelin biosynthesis genes and the pyoverdine receptor (FpvA) lead to iron-constitutive expression of the key alginate biosynthesis gene, algD, and an explicitly mucoid phenotype in both iron-limiting and iron-replete conditions. These data indicate that alginate production and mucoidy, in contrast to other types of biofilms produced by P. aeruginosa, are substantially enhanced under iron limitation. These results also have compelling implications in relation to the use of iron chelators in the treatment of P. aeruginosa CF infections. PMID:24496793

  20. Capsaicinoids improve egg production by regulating ovary nuclear transcription factors against heat stress in quail.

    Science.gov (United States)

    Sahin, N; Orhan, C; Tuzcu, M; Juturu, V; Sahin, K

    2017-04-01

    1. To examine the molecular mechanism of capsaicinoid supplementation from capsicum extract, laying Japanese quail (n = 180, 5 weeks old) were reared either at 22°C for 24 h/d (thermoneutral, TN) or at 34°C for 8 h/d (heat stress, HS) and fed on one of three diets containing 0, 25 or 50 mg of capsaicinoids per kilogram for 12 weeks (2 × 3 factorial arrangement). 2. The results revealed that exposure to HS decreased feed consumption by 10.7% and egg production by 13.6%, increased serum and ovary malondialdehyde (MDA) levels by 66.9% and 88.1%, respectively, and reduced ovary superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities by 28.3%, 48.7% and 43.8%, respectively. 3. There were magnifications in the ovary nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) levels by 42.4% and suppressions in nuclear factor (erythroid-derived 2)-like 2 (Nrf2), protein kinase B (Akt) and haem-oxygenase 1 (HO-1) levels by 29.2%, 38.2% and 30.7%, respectively, in heat-stressed quail. 4. With increasing supplemental capsaicinoids, there were linear increases in egg production, antioxidant enzyme activity, linear decreases in ovary MDA and NF-κB levels and linear increases in ovary Nrf2, Akt and HO-1 levels at a greater extent in quail reared under TN condition than those reared under HS condition. Two-way treatment interactions showed that the degree of restorations in all response variables was more notable under the HS environment than under the TN environment as supplemental capsaicinoid level was increased. 5. In conclusion, capsaicinoid supplementation alleviates oxidative stress through regulating the ovary nuclear transcription factors in heat-stressed quail.

  1. Hydrogen peroxide prevents vascular calcification induced ROS production by regulating Nrf-2 pathway.

    Science.gov (United States)

    Zhang, Wensong; Li, Yi; Ding, Hanlu; Du, Yaqin; Wang, Li

    2016-08-01

    Although vascular calcification in end-stage renal disease (ESRD) represents a ubiquitous human health problem, effective therapies with limited side effects are still lacking, and the precise mechanisms are not fully understood. The Nrf-2/ARE pathway is a pivotal to regulate anti-oxidative responses in vascular calcification upon ESRD. Although Nrf-2 plays a crucial role in atherosclerosis, pulmonary fibrosis, and brain ischemia, the effect of Nrf-2 and oxidative stress on vascular calcification in ESRD patients is still unclear. The aim of this research was to study the protective role of hydrogen peroxide in vascular calcification and the mechanism of Nrf-2 and oxidative stress on vascular calcification. Here we used the rat vascular smooth muscle cell model of β-glycerophosphate-induced calcification resembling vascular calcification in ESRD to investigate the therapeutic effect of 0.01 mM hydrogen peroxide on vascular calcification and further explores the possible underlying mechanisms. Our current report shows the in vitro role of 0.01 mM hydrogen peroxide in protecting against intracellular ROS accumulation upon vascular calcification. Both hydrogen peroxide and sulforaph